Morpholino-subunit combinatorial library and method

ABSTRACT

A method of generating a compound capable of interacting specifically with a selected macromolecular ligand is disclosed. The method involves contacting the ligand with a combinatorial library of oligomers composed of morpholino subunits with a variety of nucleobase and non-nucleobase side chains. Oligomer molecules that bind specifically to the receptor are isolated and their sequence of base moieties is determined. Also disclosed is a combinatorial library of oligomers useful in the method and novel morpholino-subunit polymer compositions.

This application is a continuation-in-part of co-owned U.S. patentapplication Ser. No. 08/015,211 filed Feb. 9, 1993, which is acontinuation-in-part of co-owned U.S. patent application Ser. No.07/988,895 filed Dec. 10, 1992 (now abandoned), which is a continuationof co-owned U.S. patent application Ser. No. 07/799,681 filed Nov. 21,1991 (now U.S. Pat. No. 5,185,444, incorporated by reference herein),which is a continuation of co-owned U.S. patent application Ser. No.07/454,057 filed Dec. 20, 1989 (now abandoned), which is acontinuation-in-part of co-owned U.S. patent application Ser. No.07/100,033 filed Sep. 23, 1987 (now U.S. Pat. No. 5,142,047,incorporated by reference herein), which is a continuation-in-part ofco-owned U.S. patent application Ser. No. 06/944,707 filed Dec. 18, 1986(now U.S. Pat. No. 5,217,866, incorporated by reference herein), whichis a continuation in part of co-owned U.S. patent application Ser. No.06/911,258 filed Sep. 24, 1986 (now abandoned), which is acontinuation-in-part of co-owned U.S. patent application Ser. No.06/712,396 filed Mar. 15, 1985 (now abandoned), and a continuation ofco-owned U.S. patent application Ser. No. 07/979,158 filed Nov. 23,1992, now U.S. Pat. No. 5,405,938, which is a continuation-in-part ofco-owned U.S. patent application Ser. No. 07/719,732 filed Feb. 9, 1993(now U.S. Pat. No. 5,166,315, incorporated by reference herein), whichis a continuation-in-part of co-owned U.S. patent application Ser. No.07/454,055 filed Dec. 20, 1989 (now U.S. Pat. No. 5,034,506,incorporated by reference herein).

FIELD OF THE INVENTION

The present invention relates to a combinatorial library formed bysequences of morpholino subunit structures, and to a method ofgenerating novel binding compounds using the library.

REFERENCES

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Eichler, J, et al., Biochemistry, 32 (41): 11035 (1993).

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Furka, A., et al., 10th International Congress on Biochemistry 5:288,Prague, Czechoslovakia, Aug. 15-19, 1988b.

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BACKGROUND OF THE INVENTION

There is current widespread interest in using combinatorial libraries ofrandom-sequence oligonucleotides, polypeptides, or synthetic oligomersto search for biologically active compounds (Kramer; Houghten,1993a-1993c, 1992, 1991; Ohlmeyer; Dooley, 1993a-1993b; Eichler;Pinella, 1993, 1992; Ecker; and Barbas). Ligands discovered by screeninglibraries of this type may be useful in mimicking or blocking naturalligands, or interfering with the naturally occurring interactions of abiological target. They can also provide a starting point for developingrelated molecules with more desirable properties, e.g., higher bindingaffinity.

Combinatorial libraries of the type useful in this general applicationmay be formed by a variety of solution-phase or solid-phase methods inwhich mixtures of different subunits are added stepwise to growingoligomers, until a desired oligomer size is reached. A library ofincreasing complexity can be formed in this manner, for example, bypooling multiple choices of reagents with each additional subunit step(Houghten, 1991; 1993c).

Alternatively, the library may be formed by solid-phase syntheticmethods in which beads containing different-sequence oligomers that formthe library are alternately mixed and separated, with one of a selectednumber of subunits being added to each group of separated beads at eachstep. An advantage of this method is that each bead contains only oneoligomer specie, allowing the beads themselves to be used for oligomerscreening (Furka, 1991; Lam, 1991, 1993; Zuckermann; Sebestyn).

Still another approach that has been suggested involves the synthesis ofa combinatorial library on spatially segregated arrays (Fodor). Thisapproach is generally limited in the number of different librarysequences that can be generated.

Since the chance of finding useful ligands increases with the size ofthe combinatorial library, it is desirable to generate librariescomposed of large numbers of different-sequence oligomers. In the caseof oligonucleotides, for example, a library having 4-base variability at8 oligomer residue positions will contain as many as 4⁸ (65,536)different sequences. In the case of a polypeptides, a library having20-amino acid variability at six residue positions will contain as manyas 20⁶ (64,000,000) different species.

Because each different-sequence specie in a large-number library maypresent in small amounts, one of the challenges in the combinatoriallibrary selection procedure is isolating and determining the sequence ofspecie(s) that have the desired binding or other selected properties.

Where the combinatorial library consists of oligonucleotides, thisproblem may be solved by amplifying the isolated sequence, e.g., bypolymerase chain reaction methods. In the case of polypeptide libraries,other methods must be employed. In one approach, where the library hasbeen formed by pooling multiple choices of reagents during synthesis, apool which is shown to have desired properties is resynthesizediteratively with lower and lower complexity until a single sequencecompound is identified.

Where the library oligomers have been formed on beads, and each beadrepresents one oligomeric specie, it may be possible to conductmicroscale sequencing on the oligomers contained on a single isolatedbead.

In another approach, the library sequences, e.g., random peptidesequences, are cosynthesized with a sequenceable tag, e.g., anoligonucleotide sequence, attached to the library sequence oligomer.That is, each oligomer in the library is associated with a distinctivesequenceable tag. Once an oligomer with the desired selection propertiesis identified, its sequence can be determined by determining thecorresponding sequence of the sequenecable tag (Brenner; Kerr).

A related approach has been to construct combinatorial libraries onbeads that are themselves tagged with distinctive tagging molecules ateach successive step in oligomer synthesis. Once an oligomer withdesired binding properties is identified, the bead to which the oligomeris attached can be "read" to identify the oligomer sequence in terms ofa sequence of tagging molecules (Ohlmeyer).

Another basic consideration in the generation of desired compounds byscreening combinatorial libraries is the nature of the selected compounditself. Polynucleotide libraries are relatively easy to generate and cansequenced at low concentrations, but have two basic disadvantages.First, the molecular variation in the library is limited by therelatively few bases that are employed, typically the standard fourbases/oligomer position. Secondly, even if an active compound isidentified, the compound may have pharmacological limitations due to itssusceptibility to nuclease digestion.

In the case of polypeptide libraries, these also can be synthesizedreadily by known solution or solid-phase methods, and the possibility of20 (or more) different side chains at each oligomer position greatlyexpands the potential variability of the library. However, as indicatedabove, screened polypeptides may be relatively difficult to sequence atthe low oligomer concentrations that are likely to be present. Further,polypeptide compounds may be susceptible to protease digestion in vivo.

Ideally, then, a combinatorial library should be easy to synthesis bystepwise solution-phase or solid-phase methods, should allow for a largenumber of different subunits at each residue position, should provide abroad range of structural diversity, and should be readily sequenceable,once a library oligomer with desired binding or other screened propertyis identified, and should be generally stable in living systems.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a combinatorial library ofnon-biological oligomers formed predominantly of morpholino subunitstructures of the form: ##STR1## where (i) the structures are linkedtogether by linkages "L" one to four atoms long joining the morpholinonitrogen of one subunit structure to the 4' cyclic carbon of an adjacentsubunit structure, and X_(i) is a purine or pyrimidine side chain, anon-nucleobase aromatic side chain, an aliphatic side chain, and/or amixed aromatic/aliphatic side chain. At least 3 of the side chains X_(i)are variable, and the library includes at least about 1,000 differentside chain sequence oligomers.

In one general embodiment, the oligomer bases in the library include acombination of nucleobase side chains, i.e., purines and/or pyrimidines,and non-nucleobase side chains, such as non-nucleobase aromatic sidechains, aliphatic side chains, and mixed aromatic/aliphatic side chains.

In another general embodiment, the oligomers are effective to hybridize,by Watson-Crick base pairing, to one of the oligonucleotide oligomers incombinatorial library of random sequence oligonucleotides.

The oligomers in the library may also have different sequences oflinkages, or be composed of the same linkages.

One preferred linkage is a 3-atom carbamate or 3-atom phosphorodiamidatelinkage.

The oligomers may also include branched structures, in which one or moreof the subunit structures forming an oligomer is linked to multiplesubunits.

The oligomers in the library may be formed on a plurality of particles,such as macroporous particles, where each particle has a surface coatingof molecules containing one of the base-sequences in the library. Theoligomer molecules are preferably attached to the particle throughcleavable linkages, e.g., chemically or photolytically cleavablelinkages.

Alternatively, or in addition, the particles may be macroreticularparticles having selected sizes in the 40-200 μm range, where theoligomers are coupled to the particles through cleavable linkages.

Alternatively, or in addition, the oligomer molecules on each particlemay represent a family of different-length oligomers having a commonsequence from one oligomer end, but different termination subunitstructures at the opposite oligomer end.

In another aspect, the invention includes a method of generating anoligomer compound capable of interacting specifically with a selectedmacromolecular ligand. The method includes contacting the receptor witha combinatorial library of oligomers of the type described above,isolating oligomer molecules that binds specifically to the receptor,and determining the sequence of bases in the isolated oligomermolecules.

Where the oligomers are designed to hybridize, by Watson-Crick basepairing, to complementary-sequence oligonucleotides in a combinatorialoligonucleotide library, the determining step includes reacting theisolated oligomers with a combinatorial library of oligonucleotides,under conditions effective to produce hybridization between the isolatedoligomer molecules and complementary-base oligonucleotides, anddetermining the sequence of the oligonucleotides hybridized to theisolated oligomer molecules.

Where the combinatorial library is formed on a plurality of particles,the particle containing the desired binding molecules is isolated, e.g.,by binding to a solid support, and oligomers on the particles are thensequenced, e.g., by release of the oligomers and micro mass spectrometryof the released oligomers.

Alternatively, particle(s) having surface-bound receptor may beidentified by reacting the particles with fluorescent-labeledanti-receptor antibodies, or by exploiting the greater density of theparticles with surface-bound receptor.

Also disclosed is a polymer composition assembled predominantly ofmorpholino subunit structures of the form: ##STR2## where (i) thestructures are linked together by linkages one to four atoms longjoining the morpholino nitrogen of one subunit structure to the 4'cyclic carbon of an adjacent subunit structure, and X_(i) is a purine orpyrimidine side chain, a non-nucleobase aromatic side chain, analiphatic side chain, and/or a mixed aromatic/aliphatic side chain.

In a related aspect, the invention includes polymer compositionassembled predominantly from morpholino subunit structures of the form:where (i) the structures are linked together by linkages one to fouratoms long joining the morpholino nitrogen of one subunit structure tothe 4' cyclic carbon of an adjacent subunit structure, and X_(i) is apurine or pyrimidine side chain, a non-nucleobase aromatic side chain,an aliphatic side chain, and/or a mixed aromatic/aliphatic side chain.

These and other objects and features of the invention will become morefully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a subunit of an oligomer formed of morpholino subunitstructures joined by linkages L;

FIG. 2 shows a morpholino subunit structure used in the FIG. 1 oligomer;

FIG. 3 illustrates the conversion of a ribonucleotide to a morpholinosubunit structure;

FIG. 4 shows the conversion of glucose to a morpholino subunitstructure;

FIGS. 5A-5E shows representative X_(i) purine and pyrimidine nucleobaseside chains (3A), modified nucleobase side chains (3B); aromatic sidechains (3C), aliphatic side chains (3D, and mixed aromatic/aliphaticside chains (3E), where the X_(i) side chains are shown attached to the1' morpholino ring position in FIG. 1;

FIG. 6 shows stereochemical options for X_(i) and linkage atoms Y in amorpholino subunit structure;

FIG. 7 illustrates a variety of activated subunit structures useful informing the oligomers of the invention;

FIG. 8 illustrate compounds which may be converted to morpholino subunitstructures during oligomer assembly;

FIGS. 9A and 9B illustrate methods for the conversion of ribose andglucose compounds, respectively, to morpholino subunit structures duringoligomer assembly;

FIG. 10 illustrates orientation about the X_(i) bond in morpholinosubunit structures;

FIG. 11 illustrates rotational freedom about the amide linkage inselected tertiary amine linkages;

FIGS. 12A and 12B show representative one-atom linkages in oligomers ofthe invention;

FIGS. 13A and 13B illustrates the syntheses of oligomers having one-atomlinkages between morpholino subunit structures;

FIG. 14 shows representative two-atom linkages in oligomers of theinvention;

FIG. 15 shows representative three-atom linkages in oligomers of theinvention;

FIG. 16 shows representative four-atom linkages in oligomers of theinvention;

FIG. 17 shows a portion of a branched oligomer formed in accordance withanother embodiment of the inventions;

FIG. 18 illustrates the synthesis of the branched portion of theoligomer shown in FIG. 17;

FIGS. 19A-19C show three different cleavable linkers used in attaching amorpholino oligomer to a tether bound to a particle surface;

FIG. 20 shows a portion of a bead having a surface coating ofsame-sequence oligomer molecules;

FIG. 21 illustrates a portion of a bead surface having a family ofsame-sequence, different-length oligomer molecules;

FIG. 22 illustrates a solid-phase method for isolating a particlecarrying a surface coating of oligomer molecules that bind to a receptorattached to a solid support; and

FIGS. 23A-23D illustrate various solution-phase methods for selectingparticles carrying a surface coating of oligomer molecules that bind toa receptor attached to a solid support (23A), by first binding receptorto a particle having the desired oligomer sequence (23B), andidentifying particle(s) having bound receptor by further reacting theparticle with fluorescent-labeled anti-receptor antibody (23C), or byseparating particle with bound receptor on the basis of its increaseddensity (23D).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise stated, the terms below have the following meanings:

A "morpholino subunit structure" refers to a morpholino structure of theform: ##STR3## where X_(i) is a side chain.

A "side chain" refers to one of several different X-groups that may becarried on a morpholino subunit structure.

A "subunit" in an oligomer includes a subunit structure and one of itsassociated linkages to an adjacent subunit structure. That is, theoligomer is composed of linked subunits, which in turn are composed ofsubunit structures and associated linkages.

An "oligomer" refers to a polymer composed of typically between about4-15 subunits. The oligomers of the present inventions are formed ofmorpholino subunit structures that are linked together by linkages ofone to four atoms long joining the morpholino nitrogen of one subunitstructure to the 4' cyclic carbon of an adjacent subunit structure.Although morpholino subunit structures are the predominant subunit formin the oligomers, other subunit structures may also be employed.

The "sequence of side chains" in an oligomer refers to the sequence ofindividual side chains on successive subunit structures in an oligomer,on progressing from one end of the oligomer to the other.

The "sequence of linkages" in an oligomer refers to the sequence ofindividual linkages linking successive subunit structures in anoligomer, on progressing from one end of the oligomer to the other.

A "combinatorial library of oligomers" refers to a library of oligomermolecules containing a large number, typically between 10³ and 10⁷different-sequence oligomers, typically defined by a different sequenceof side chains, or a combination of different sequences of side chainsand linkages. Each sequence in a library is preferably represented by aplurality, e.g., 10¹⁰ -10¹² molecules of the same sequence.

A "branched oligomer" refers to an oligomer having one or moremorpholino subunit structures that are covalently attached to a linkagethat itself directly links two additional morpholino subunit structuresin the oligomer. The sequence of side chains in a branched oligomerrefers to the sequence of side chains in the longest chain of theoligomer, with branched side chains being indicated in parenthesis atthe linkage position to which the branch is joined. Thus, an oligomersequence of the form: X₁ X₂ X₃ X₄ (X₄₋₁ X₄₋₂)X₅ X₆ refers to an eightmerhaving the linear sequence of sidchains X₁ X₂ X₃ X₄ X₅ X₆, and abranched sequence of side chains X₄₋₁ X₄₋₂ on a two-subunit chainattached to the linkage between the fourth and fifth subunit structuresin the linear chain.

A "nucleobase" side chain is a purine or pyrimidine side chain attachedto the morpholino moiety through the N9 of the purine or the N1 of thepyrimidine.

A "non-nucleobase aromatic" side chain is a substituted or unsubstitutedaromatic side chain that is not a purine or pyrimidine.

An "aliphatic" side chain refers to a side chain having the generalstructure --(CH₂)_(m) --X where m=1-5 and X is H, an unbranched orbranched alkane, alkene or alkyne, OH, SH, an amine, a halide, analdehyde, an acid, an amide, or an ester group.

A "mixed aromatic/aliphatic" side chain is an aromatic side chainsubstituted with an aliphatic side chain.

A "receptor" is a macromolecule capable of specifically interacting withligand molecule, including oligomers of the inventions. Binding of theligand to the receptor is typically characterized by a high bindingaffinity i.e., K_(m) >10⁵ and is intended to affect, e.g., inhibit, thefunction of the receptor in its normal biological setting. The receptoris also referred to herein as a target structure.

II. Morpholino Subunit Structures

The invention includes, in one aspect, a combinatorial library ofoligomers having the general form shown in FIG. 1. The oligomers areformed from morpholino subunit structures of the form shown in FIG. 2,where the subunit structures are linked together by linkages L one tofour atoms long joining the morpholino nitrogen of one subunit structureto the 4' cyclic carbon of an adjacent subunit structure. The X_(i)groups or side chains in the oligomers are nucleobase or non-nucleobaseX groups, as will be described below.

Each morpholino subunit structure contains a morpholino backbone moiety,which allows linking the subunit structure to other subunit structuresin a defined order, and a side chain X^(i). These morpholino subunitstructures have the general structure shown in FIG. 2, where X^(i), theside chain, is hydrogen or an organic substituent, which may be in aprotected form; Y, which may be in an activated or protected form, is agroup which allows coupling of the morpholino subunit to the morpholinonitrogen or Z group of another morpholino subunit, or other structure; Zis hydrogen, a protective group, or other group, which may be in anactivated or protected form, which is suitable for coupling to the Ygroup of another morpholino subunit or other structure; and, X and Ysubstituents have defined stereochemical orientations.

A. Preparing Subunit Structures

Morpholino subunit structures can be prepared from ribonucleosides andrelated substituted furanosides, as illustrated in FIG. 3 and describedin Example 1, and from substituted glucose and related hexopyranosides,as illustrated in FIG. 4 and described in Example 2.

A variety of side chains, X, contribute to the structural diversityachievable with this class of oligomers, which in turn facilitates theselection of oligomer species with desirable biological activities.FIGS. 5A-5E illustrate representative types of side chains of morpholinosubunits which can be prepared from natural products and simple chemicalreagents. FIG. 5A shows representative nucleobase side chains. Example 3describes synthetic routes to such structures.

FIG. 5B shows a number of modified nucleobase side chains modified byaddition of R groups at various ring positions, as indicated. Here R ispreferably an aliphatic group, such as methyl. Example 4 describessynthetic routes to nucleobases modified at one or more such sites.

FIG. 5C shows representative, aromatic, non-nucleobase side chains. HereX is OH or OR, where R is a lower alkyl, or a primary, secondary ortertiary amine. Y and Z may be any of a variety of small groups, such asCN, halogen, NO₂, OH, alkoxy, aldehyde, and amine groups. Examples 5A-5Edisclose methods for preparing morpholino subunit structures withexemplary non-nucleobase aromatic side chains.

Also contemplated are morpholino subunit structures with aliphatic sidechains, as shown in FIG. 5D, where the R groups may be branched orunbranched alkanes, alkenes, or alkynes. Exemplary morpholino subunitshaving these types of side chains are described in Examples 5F-5I.Finally, the side chains may be mixed aromatic/aliphatic groups, such asshown in FIG. 5E.

B. Stereochemical Control of X and Y

The stereochemistry of the X side chain about the 1' carbon of themorpholino moiety can be selected to be either alpha or beta, and thestereochemistry of the Y group about the 4' carbon of the morpholinomoiety can also be selected to be either alpha or beta, as illustratedin FIG. 6. Basic synthetic strategies for achieving these selectedstereochemical options are illustrated in Example 6.

Morpholino oligomers are assembled predominantly by linking the Y groupof one morpholino subunit structure to the morpholino nitrogen or Zgroup of another morpholino subunit structure (FIG. 2), where one ofthese groups is nucleophilic and the other is electrophilic. Theresulting intersubunit linkage, L, should be stable to conditions ofsynthesis and any required deprotection steps, as well as stable underthe conditions of use.

A preferred assembly method is to use subunit structures in which thenucleophilic moiety is in a protected form (often protected with atrityl group), and the electrophilic moiety is in an activated form, oris activated in situ just before or during the coupling step. FIG. 7illustrates a number of representative morpholino subunits so configuredfor oligomer assembly, and Example 7 describes their preparation.

C. Conversion of Subunits to Morpholino Subunit Structures duringoligomer assembly.

In addition to oligomer assembly by coupling of preformed morpholinosubunit structures, morpholino oligomers can also be assembled by amethod in which the morpholino backbone moieties are formed in thecourse of oligomer assembly. FIG. 8 illustrates a number ofrepresentative compounds suitable for this purpose, and Example 8describes their synthesis. Key structural characteristics of thebackbone moiety of any such compound include a primary aliphatic aminemoiety and two or more vicinal hydroxyls.

II. Oligomer Assembly

This section describes methods for preparing morpholino oligomers of thetype used in the invention, and the spatial and geometric considerationsimportant in polymer construction.

A. Side Chain Rotational Freedom

The diversity of spatial arrangements of the side chains in a library ofmorpholino oligomers can be increased appreciably by use of nucleobaseand similar side chains which are structured so as to control theirorientation about the bond between the side chain and the 1' atom of themorpholino backbone moiety. Based on NMR studies conducted in support ofthe invention, the morpholino backbone moiety exists predominantly in achair conformation, with the X and Y groups positioned equatorial.

As indicated in FIG. 10, pyrimidines and related side chains whichcontain a bulky group at the 2 position and a hydrogen at the 6 positionexist almost exclusively in the anti conformation about the X-1' bond.In contrast, purines and related side chains which contain a hydrogen atthe 8 position can exist in either the syn or anti conformation aboutthe X-1' bond. Further, purines and related side chains which contain abulky group at the 8 position exist predominantly in the synconformation about the X-1' bond.

B. Oligomer Linkage Rotomers

In selecting suitable intersubunit linkage types, a key objective is toprepare an oligomer library containing a collection of molecules, eachof which has a definable spatial arrangement of side chains. In thisregard, certain linkage constraints need to be considered. For example,if a bond has a high barrier to rotation, resulting in two distinctrotomers, and both rotomers are present at significant concentrations,then a given molecular specie containing such linkages would be expectedto contain 2^(n) distinct, but slowly interconverting conformations,where n is the number of rotomer-generating restricted-rotation bonds inthat oligomer. This results in a diverse collection of rotomers, onlyone of which has the desired spatial arrangement of side chains.

Even if the desired rotomer can be isolated, except in special cases itwill slowly interconvert to form the original mixture of rotomers. Inthis context, tertiary amides and related groups containing a carbonyllinked to a nitrogen containing two alkyl groups are well known toexhibit two distinct rotomer forms which interconvert only very slowlyat physiological temperatures. For instance, amides containing thedialkyl nitrogen of proline have been reported to have a T_(1/2) ofrotation of many hours at 37° C., and the temperature of coalescence(Tc), determined by nuclear magnetic resonance, has been found to be114° C. To illustrate the impact of such rotomers on the spatialarrangement of side chains in polymeric structures, it is the slowinterconversion of the tertiary amide of proline in polypeptides whichis largely responsible for the failure of many proteins to spontaneouslyrenature after heat denaturation.

In view of the above considerations, intersubunit linkages containingtertiary amides might seem to be undesirable for morpholino oligomersdestined for biological applications. However, in experiments carriedout in support of the present invention, it has been discovered that inthe special case of a carbonyl linked to the ring nitrogen of morpholinosubunits, there is a relatively low barrier to rotation about thisparticular tertiary amide linkage, evidenced by the low Tc values forthe amide-linked morpholino structures shown in FIG. 11.

Accordingly, tertiary amide intersubunit linkages to the ring nitrogenof the morpholino backbone moiety are now known to be acceptablelinkages for a variety of morpholino oligomers. Novel intersubunitlinkages of this type, which heretofore appeared to be unacceptable onthe basis of previously available information, are illustrated instructure 1 of FIG. 12, structure 4 of FIG. 14, structure 2 of FIG. 15,and structure 2 of FIG. 16.

C. Forming Oligomers with One-Atom Linkages

One-atom-length linkages between morpholino subunits afford oligomerstructures with little conformational freedom, which, in turn, minimizesthe entropy cost of binding between such oligomers and suitable targetstructures. FIG. 12 shows two one-atom-length intersubunit linkages.

Methods for joining morpholino subunits to form these one-atom-lengthintersubunit linkages are illustrated in FIG. 13 and described inExample 9. Alternatively, oligomers containing one-atom linkages may beconstructed by converting the last-added subunit to a morpholino groupduring oligomer synthesis, as illustrated in FIG. 9 for ribose andglucose subunits. The methods of synthesis are detailed in Example 10.

D. Forming Oligomers with Two-Atom Linkages

Two-atom-length linkages afford oligomer structures with greaterconformational freedom than those with one-atom linkages. Exemplarytwo-atom linkages, shown in FIG. 14, can be formed by the generalmethods illustrated in FIG. 13 and described in Example 9, or by thegeneral method illustrated in FIG. 9 and described Example 10.

Additional methods of synthesis of oligomers from morpholino subunitsare also described in co-owned U.S. Pat. Nos. 5,235,033, and 5,185,444,which are incorporated herein by reference.

E. Forming Oligomers with Three-Atom Linkages

FIG. 15 illustrates and Example 11 describes methods of formingrepresentative three-atom-length linkages between morpholino subunits.Additional methods are described in co-owned U.S. Pat. Nos. 5,235,033and 5,185,444. Such linkages properly space and position suitablenucleobase side chains for Watson/Crick binding to complementarysingle-stranded oligonucleotides, and to suitable complementarymorpholino oligomers. Further, incorporation of a relatively rigidcarbamate or thiocarbamate intersubunit linkage (structure 1 of FIG. 15)largely precludes stacking of adjacent nucleobase side chains in aqueoussolution, resulting in substantial hydrophobic character for suchnucleobase-containing oligomers. In contrast, incorporation ofrelatively flexible sulfonyl, and particularly phosphoryl linkages(structures 3, 4, and 5 of FIG. 15) affords good stacking of adjacentnucleobase side chains in aqueous solution, resulting in generally goodwater solubility for many such nucleobase-containing oligomers.

F. Forming Oligomers with Four-Atom Linkages

Still greater spacing of the side chains in a morpholino oligomer isafforded by four-atom-length linkages between subunits. Representativefour-atom-length intersubunit linkages are shown in FIG. 16. Methods offorming such linkages are described in Example 12.

It will be appreciated that non-morpholino subunits can be introducedinto the morpholino-subunit oligomers, either in linear or branchedportions thereof, by selecting subunit structures having suitable donorand acceptor groups, and incorporating these subunits into the oligomerby the general coupling methods described herein.

G. Forming Oligomers with Branches

Utilization of one or more branches in an oligomer can substantiallyincrease the spatial diversity of its side chains relative to unbranchedoligomers. One or more branches in an oligomer also serves to increasestructural complexity by positioning a greater number of side chains ina small area, resulting in an increased likelihood of multipleinteractions with a suitable target structure. FIG. 17 shows a portionof a morpholino-subunit oligomer having a branch linkage at which a 1-Nsubunit branch extends from a dominant linear portion of the oligomer(the longest linear chain in the oligomer).

The branch oligomers may be formed by a variety of methods, typically byemploying a branched structure which provides two nitrogens, each ofwhich can be reacted with an activated subunit, as illustrated in FIG.18. Example 13 describes the preparation of several such in-line typebranches. Example 14 describes the synthesis of branced oligomers withhub branches. Example 15 describes the covalent joining of branchedends.

IV. Oligomer Libraries

This section describes the preparation and properties of combinatoriallibraries of oligomers of the type described above. In general thelibraries are constructed to contain oligomers having a large number ofdifferent sidechain sequences and, optionally, linkage sequences.Preferably, the oligomers making up the library include subunitstructures with at least three, and typically 5-20 different sidechains, and at least about 1,000 different side chain sequences.Preferably the library contains 10⁴ to 10⁷ different sequences, whichmay include different sidechains and different linkages. Eachdifferent-sequence specie in the library preferably exists in multiplecopies, preferably 10¹⁰ or more where microsequencing is employed todetermine oligomer sequence.

Subunits with the same X side chain but different Y and/or Z groupsconstitute different subunits, since changing the Y or Z moietygenerally alters the relative spatial arrangement of the side chain inthe oligomer. Further, subunits with the same X side chain and the sameY and Z, but with differing stereochemistry about the X and/or Y groupsalso constitute different subunits since changing the stereochemistrygenerally affords a substantial alteration of the spatial arrangement ofthe side chain in the oligomer. As a consequence, hundreds of differentsubunits can be readily prepared, in contrast to the four nucleotidesubunits of enzymatically-prepared oligonucleotides and the 20 aminoacids of biologically-generated peptides.

Combinatorial libraries of the type used in the invention may be formedby a variety of solution-phase or solid-phase methods in which subunitsare added stepwise to growing oligomers, until a desired oligomer sizeis reached, as outlined below.

B. Solid-Phase Particle Library

In one preferred method, the library is formed by solid-phase syntheticmethods in which beads containing different-sequence oligomers that formthe library are alternately mixed and separated, with one of a selectednumber of subunits being added to each group of separated beads at eachstep. Each bead in the resultant library contains only one oligomerspecie, allowing a single bead, once identified as containing thedesired binding oligomer sequence, to provide oligomer for sequenceidentification.

One preferred particle or bead for use in library construction is amacroporous bead having a density of between 1 and 1.3, and a size ofabout 20-200 μm. With reference to FIG. 20, the particle, which is shownfragmentarily at 30, is preferably derivatized with a separator chain ortether, such as tether 32, having a cleavable linkage 34 adjacent itsdistal or free end, such as detailed in Example 16.

Highly crosslinked macroporous polystyrene particles (buoyant density1.05 g/cm³) are particularly suited for use in preparing sucholigomer-particles, and commercially available polystyrene particleswith amine, hydroxyl, or carboxyl moieties covering their surfacesprovide suitable sites for linking tethers and dyes or fluorescentgroups.

Polyethylene glycols and polypropylene glycols, preferably with averagemolecular weights in the range of 400 to 6000, serve as effectivetethers. Use of higher molecular weight tethers (>1000 MW) typicallyafford oligomer-particles with higher target binding capacities in thetypical case where the target structure is relatively large, for exampleproteins, which generally range from 30 to 100 angstroms in diameter.

The selectively cleavable anchor between the tether and the oligomershould be stable to conditions used for subunit coupling, deprotectionof termini, deprotection of side chains, and the aqueous conditions usedfor assessment of target binding. The linker should also be easily andselectively cleavable under simple conditions. Three linkages whichsatisfy these criteria are: disulfide (cleavable with mercaptoethanol);derivatives of 4-hydroxymethyl-3-nitro-benzoic acid (cleavable with 350nm light); and vicinal alcohols (cleavable with periodate), asillustrated in FIGS. 19A-19C.

Macroporous particles, each containing many femptomoles to a fewpicomoles of functional sites on its surfaces, and preferably having abuoyant density appreciably less than that of the target structure, arereacted to add to the surface a water soluble tether ending in aselectively cleavable anchor. Remaining sites on the particle surfacesmay be reacted with a suitable dye or fluorescent group, or dye orfluorescent material may be incorporated within the particle matrixduring its polymerization. Such label is desirable to improvevisualization of the oligomer-particles in the course of assessing fortarget binding.

A preparation of particles, containing at least several times as manyparticles as there will be oligomer species in the library, is nextdistributed into equal portions, where the number of portions istypically the same as the number of different subunits in the set ofsubunits to be used for assembly of the variable portion of theoligomers in the oligomer library. Each portion of particles is thanreacted with a different subunit of the subunit set, such as subunitstructure 36 in FIG. 20. After coupling, all portions of particles arecombined, mixed thoroughly, washed, and treated to deprotect theoligomer termini.

This subunit addition cycle, comprising distribution of particles intoseparate portions, coupling each portion with a different subunit,recombining, mixing, washing, and deprotection of the oligomer termini,is repeated until the desired number of subunits have been added to givea complete library of oligomers covalently bound to the particles.

The collection of oligomer-particles are next treated to removeprotective groups on the side chains, and then washed, after which theyare ready for use. Example 16 describes representative procedures forpreparing such oligomer-particles.

One particle in a completed library is illustrated in FIG. 20. Theparticle, shown fragmentarily at 38, contains a plurality of oligomermolecules, such as molecules 40, each having the oligomer sequenceABCDEFGH, representing the sequence of eight different subunit sidechains. As shown, each oligomer molecules is attached to the particlethrough a tether, such as tether 42, containing a cleavable linker, suchas linker 44. Methods for forming library beads of the type justdescribed are given in Example 16.

C. Preparing Oligomer Sequence Families

In many applications, it may be desirable to prepare a library ofoligomer families, where each family consists of different-length, butsame-sequence oligomer molecules attached to the same particle. That is,the molecules all have the same sequence, beginning from one oligomerend, but contain different numbers of subunits, typically includingmolecules that contain from 1 to N subunits, where the largest oligomercontains N subunits.

One exemplary particle in a completed library is illustrated in FIG. 21.The particle, shown fragmentarily at 46, contains a plurality ofoligomer molecules, such as molecules 48, each having a portion of thesequence ABCDEFGH (including some molecules containing the entiresequence ABCDEFGH), of an oligomer having this side chain sequence. Asabove, the oligomer molecules are each attached to the particle througha tether, such as tether 50, containing a cleavable linkage, such aslinkage 52. Two general methods for forming bead libraries of the typejust will now be described and are detailed in Example 17.

In one method, when coupling a given subunit with a particular portionof particles a mixture of activated subunits is used wherein a definedfraction in the mixture (50% to 90%) contains a protective group which,after coupling, can be cleaved to allow coupling of a subsequent subunitin the next subunit addition cycle. The remaining fraction of subunit inthe mixture (10% to 50%) is capped with a group which precludes couplingin subsequent subunit addition cycles. By this method eacholigomer-particle will contain a family of oligomer species, this familycomprising capped oligomers ranging from 1 to N-1 subunits, and anuncapped oligomer of N subunits.

If it is desirable to have the cap present during subsequent use ofthese particles for assessment of target binding, an acetyl cap isconvenient. If it is desirable to remove the cap prior to use in thetarget binding assessment, a trifluoroacetate cap can be used. Thisgroup is removed during the treatment with ammonium hydroxide typicallyused for deprotecting side chains.

In using this method of generating families of oligomers on a singleparticle, when branched oligomers are prepared, the method can lead toambiguities in sequence information generated in the final mass spectralanalysis. Such ambiguities arise because truncation can occurindependently in each of the branches. A strategy to remove most, andoften all of this sequence ambiguity is to incorporate a dual-mass capin those oligomers which are truncated in the first-synthesized branch.One representative dual-mass scheme which is easily assessed in the massspectral sequencing step entails utilizing a mixture of acetyl andbenzoyl capped subunits in the subunit mixtures used for thefirst-synthesized branch of a branched oligomer, while using just theacetyl cap for subunit mixtures used for all other subunits additions.This affords a mass series in which eah oligomer truncated in thefirst-synthesized branch is distinguished by dual masses, separated by62 mass units. Another dual-mass scheme entails using a mixture ofacetyl and trifluoroacetyl capped subunits for the first-synthesizedbranch, and just trifluoroacetyl capped subunits for subunit mixturesused for all other subunit additions. After assembly of the oligomerlibrary is completed, the trifluoroacetyl moieties are cleaved bytreatment with ammonia. This affords a mass series in which eacholigomer truncated in the first-synthesized branch is distinguished bydual masses, separated by 42 mass units.

An alternative method for generating oligomer-particles containingfamilies of different-length, same sequence oligomers has been developedfor morpholino oligomers. In this approach (which does not utilizemixtures of capped and protected subunits), the protective group on thechain termini which is to be cleaved at the end of each subunit additioncycle is a trityl group on the ring nitrogen of a morpholino subunit.Trityl cleavage is carried out with dichloromethane solutions whichcontain >5% formic acid (v/v). This formic acid treatment is effectiveto formylate the terminal morpholino nitrogen at a relatively constantrate for a given concentration of formic acid. For example, 7% by volformic acid in dichloromethane formylates morpholino chain termini at arate of 2.5%/hr at 20° C.

Therefore, by simply exposing the particles containing nascentmorpholino oligomers to a suitable formic acid deprotection solution fora selected period of time one can achieve truncation of a desiredpercent of chain termini. When concentrated ammonium hydroxide, neat oras a 1:1 v/v mixture with dimethylformamide, is used for deprotectingthe side chains, these formyl moieties are cleaved from the truncatedchains, leaving the terminal morpholino nitrogen in the free base form.

For branched oligomers, it is desirable to utilize the dual mass cappingstrategy described above for the first-synthesized branch, and thiscontrolled formylation method for all other subunit additions.

V. Selecting Specific-Sequence Oligomer Molecules

The combinatorial libraries described above are used to select one ormore oligomer species in the library that demonstrate a specificinteraction with a selected receptor. The receptor is any biologicalreceptor of interest, that is, one for which it is desired to identify aoligomer (ligand) that binds specifically to the receptor, to affect thefunctioning of the receptor in its normal physiological setting.

For example, the receptor may be an enzyme, where the oligomer is ableto bind to the active site of the enzyme or otherwise inhibit the actionof the enzyme on a normal substrate.

In another general embodiment, the receptor may be a cell receptorprotein, such as an ion channel or other transport receptor protein, ora receptor site for a hormone or other cell effector, or a receptor sitefor binding of pathogenic bacteria or viruses to a cell surface. Thereceptor protein may be associated with isolated cells with culturecells, with biological membrane particles isolated from tissues, withcells which are transformed to produce the receptor recombinantly, orwith isolated cell receptors. Receptor proteins of this type, andexpressed or isolated in a variety of forms, have been described in theliterature, such as that cited above.

In a related embodiment, the receptor is an antibody or antibodyfragment, where it is desired to identify an "artificial" epitope ligandthat binds specifically and with high affinity to the antibody.

In a typical application, the library of oligomers is screened foroligomer (ligand) molecules that bind specifically and with highaffinity, e.g., with a binding constant K_(B) greater than 10⁶ M, to thereceptor. In one embodiment, illustrated in FIG. 22, receptor molecules,such as molecules 54, are attached to a solid support 56. Attachment maybe by way of covalent or noncovalent attachment of an isolated receptorto the surface. Alternatively, the solid surface may be cells havingsurface-bound receptor, or the cells themselves may be anchored on asolid support. Methods for attaching proteins or cells to a solidsupport are well known.

In the selection method the support is contacted with the libraryoligomers, i.e., the different-sequence oligomer molecules making up theoligomer library, under conditions that allow binding of only one or afew oligomer species to the receptor. The binding conditions, e.g., saltconcentration, pH and/or temperature may be selectively varied,according to standard methods, to ensure that only the highest-affinityoligomer species are bound to the receptor.

In the method illustrated in FIG. 22, the library is constructed asabove to include a library of particles, each containing multiple copiesof the same-sequence oligomer. The particles are reacted withsupport-bound receptors under conditions which promote binding to thesolid surface of library particles, indicated at 58, that carryhigh-affinity ligands, such as oligomers 60, for the receptor.

Following this binding, the solid surface is washed to remove unbound orless tightly bound particles, and the one or more remaining boundparticles are then analysed, according to methods described below, todetermine the sequence of the high-affinity oligomers.

FIGS. 22A-22D illustrate various solution-phase methods for identifyingdesired library oligomer sequence(s). Here a library particle 62carrying molecules, such as molecules 64, having one of the librarysequences is reacted with the receptor 66 in solution phase, underconditions which lead to receptor binding to high-affinity libraryparticles, as illustrated in FIG. 23B.

The particles with bound receptor may be further reacted withreported-labeled antibody 68 specific against the receptor molecules, tolabel the desired library particle(s) with a suitable reporter, such asa fluorescent label, as indicated in FIG. 23C. The labeled particles maybe removed by micromanipulation, e.g., under fluorescent microscopy, orusing standard cell sorting methods to isolated reporter-labeledparticles.

Alternatively, the particle density may be so selected that binding ofreceptor protein to the particles increases the particle densitysufficiently to separate receptor-bound particles on the basis ofdifferential density, as illustrated in FIG. 23D. The figure shows areceptor-bound particle being separated by centrifugation or particlesettling in a medium 70 whose density is intermediate between thedensity of particles 72 that do not contain bond receptor, and those,such as particle 62, that do.

A preferred type of particle for density separation which has desireddensity and solvent-resistance properties is macroporous polystyreneparticles in the size range of 20 to 200 microns in diameter. Suchmacroporous particles, which are used for ion exchange chromatography,can be obtained which have large surface to mass ratios, suitable poresizes (in the range of 400 to 1000 angstroms), and which have surfacescontaining covalently linked amine, hydroxyl, or carboxyl groups, whichprovide convenient sites for anchoring oligomers.

Other methods of isolating library particles having desiredreceptor-binding properties are also contemplated. For example, in thecase of an antibody receptor, the bivalent nature of the antibody couldbe used to crosslink particles having high-binding ligands on theirsurfaces.

Alternatively, where the receptor is carried on cell surface, thelibrary particles, such as polymeric particles having particle sizes inthe 0.5-2 μm range, are reacted with the cells under conditions thatpromote ligand-specific receptor surface binding, followed by one ormore cell washes, to remove unbound particles, and release of boundparticle(s) from teh washed cells.

VI. Determination of Oligomer Sequence

Once a library oligomer having a desired interaction, e.g., bindinginteraction, with a receptor is identified, the oligomer molecules, itmust be sequenced to determine the sequence of side chains. This may bedone in accordance with various sequencing methods, several of which aregiven below, and in Example 18.

A. Oligonucleotide Amplification

As indicated above, the oligomer library may be formed of oligomermolecules having (i) nucleobase side chains and (ii) intersubunitlinkages that allow Watson-Crick base pairing between the nucleobasesthe bases of complementary-base sequence oligonucleotides.

In this embodiment, the isolated oligomer molecules, whether in solutionphase or carried on a particle, are reacted with a combinatorial libraryof oligonucleotides, under hybridization conditions that permitcomplementary strand hybridization between the selected oligomermolecules and same-sequence oligonucleotide molecules.

The bound oligonucleotide molecules are then released, made doublestranded, amplified, e.g., by polymerase chain reaction, and sequencedaccording to standard methods. The sequence obtained corresponds then tothe side-chain sequence of the isolated oligomer molecules.

Both the library oligomer molecules and the random-sequenceoligonucleotides may have known-sequence oligonucleotide end segments toenhance hybridization between the two. If the oligomers are designed tocontain a mixture of nucleobases and either modified nucleobases ornon-nucleobase sidechains, the stringency of the hybridizationconditions may be reduced, to allow some non-pairing with oligomerbases. Sequencing the bound oligonucleotides would be effective toreconstruct the oligomer sequence in some, but not all, subunitpositions.

B. Isolated Particle Sequencing

In the embodiment in which the library oligomers are contained onparticles, with each particle containing only one oligomer sequence, theisolated particles are treated to release the attached oligomermolecules, and the release molecules are sequenced, e.g., by micro massspectrometry, such as detailed in Example 17 below. Preferably, eachparticle provides sufficient oligomer material for microsequencing, toavoid the problem of sequencing mixed-sequence oligomers derived fromdifferent beads.

In a modification of this approach, the library particles are preparedto contain a family of different-length, same-sequence oligomermolecules, as described above. After cleavage of the family of oligomersfrom an isolated particle surface, sinapinic acid is added and thematerial is placed under reduced pressure to remove volatile material,and then inserted into a mass spectrometer, preferably alaser-desorption time-or-flight mass spectrometer. By this means, whenthe oligomer-particles are assembled as described above, the massspectrum permits ready determination of the exact mass of each subunit,as well as the order of said subunits in each oligomer specie of thefamily of oligomers. This procedure even affords exact structures forproperly assembled branched oligomers.

Once the sequence has been determined for the family of N oligomersspecies from a single particle, oligomers having that sequence, andsizes ranges from 1 to N subunits, may be prepared to determine theoligomer length that affords highest binding affinity, or which providesthe best compromise between high binding affinity and length.

C. Solution-Phase Iterative-Search Method

In those cases where it is desirable to test oligomers free in solution,instead of attached to the surface of a particle, oligomer libraries canbe prepared as described earlier, but cleaved from the support beforetesting. When testing is carried out with oligomer free in solution, onecan assess for a broader range of activities than just target binding,such as inhibition or activation of enzymes, blocking of binding ofligands, etc.

Screening of these oligomers free in solution, along with an iterativeselection and synthesis process for the systematic identification ofoligomers having a desired biological activity can be carried out bymethods modeled after those reported by Houghten, et al. (Nature 354 84(1991)).

From the foregoing, it can be appreciated how various objects andfeatures of the invention are met. The combinatorial library is easy tosynthesis by stepwise solution-phase or solid-phase methods, with themorpholino subunit structures making up the oligomers being preformed orformed during stepwise synthesis. The ability to construct subunitstructures with a wide range or nucleobase, modified nucleobase,aromatic, aliphatic, and mixed base side chains allows the constructionof libraries having virtually any desired degree of complexity. Thepossible complexity of the libraries is further enhanced by thestereochemical variations, and variations in linkages that are possible,as well as the ability to construct branched oligomers.

The oligomers may be readily screened for a desired interaction with aselected receptor, e.g., according to binding affinity. The inventionprovides a variety of methods for isolating library particles containingdesired oligomer ligands, as well as simple methods for determiningoligomer sequences.

The following examples illustrate various synthetic procedures forpreparing oligomers useful in the invention. The examples are intendedto illustrate, but not limit the scope of the invention.

Materials

Unless otherwise indicated, chemicals are purchased from AldrichChemical Co. Abbreviations used: Tr=trityl (triphenylmethyl);DMT=4,4'-dimethoxytrityl; CBz=(phenylmethyl)oxycarbonyl;Boc=(1,1-dimethyl)ethyloxycarbonyl; FMOC=(9-fluorenylmethyl)oxycarbonyl;TBDMS=tert-butyldimethylsilyl; TBDPS=tert-butyldiphenylsilyl,Ac=acetate; Bz=benzoate, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene,NMP=1-methyl-2-pyrolidinone.

Referenced Methods

The following references disclose various synthetic procedures referredto in the examples, and are incorporated herein by reference:

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Araki, et al., Tetrahedron Lett. 29:351 (1988).

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Bellosta and Czernecki, J. Chem. Soc., Chem. Commun., 199 (1989).

Benseler and McLaughlin, Synthesis, 46 (1986).

Carpino, et al., J. Chem Soc., Chem. Commun. 358 (1978).

Carpino and Han, J. Org. Chem. 37:3404 (1972).

Chow and Danishefsky, J. Org. Chem. 55:4211 (1990).

Cook, et al., J. Amer. Chem. Soc., 98:1492 (1976)

Czernecki and Ville, J. Org. Chem. 54:610 (1989).

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Yanagisawa and Kanazaki, Heterocycles, 35:105 (1993).

EXAMPLE 1 Preparation of Morpholino Subunits from SubstitutedRibofuranosides

A. General procedure for protection of the R group.

1. Protection of amino groups on heteroaromatic rings. Theribofuranoside (1 mol) is suspended in acetonitrile (2.5 L) andhexamethyldisilazane (5 mol) is added. The solution is refluxed untilsolution is complete and the solvents then are removed by distillation.Residual hexamethyldisilazane is removed by addition of xylene (1L) andremoval by distillation. The residue is dissolved in pyridine (2.5L) andtreated with trimethylchlorosilane (5 mol). After the solution isstirred for 15 minutes, an acid chloride or chloroformate or similaracylating agent (5 mol) is added and the solution is maintained at roomtemperature for 3-24 hours. The reaction is cooled in an ice bath andwater (500 ml) is added. After stirring for 5 minutes conc. ammonia (500ml) is added, and the reaction is stirred for 15 minutes. The solutionis evaporated to near dryness and the residue is poured into water (10L). The product is isolated by filtration.

Representative examples of ribofuransoides and the correspondingacylating agents are cytidine and benzoyl chloride; adenosine andbenzoyl chloride; guanosine and the ester formed from1-hydroxybenzotriazole and phenylacetyl chloride (Benseler andMcLaughlin). Alternatively, the FMOC group may be introduced by themethod of Heikkila and Chattopadhyaya.

2. Protection of aliphatic or carbocyclic aromatic amines. Amino groupsare protected by conversion to the trifluoroacetamide by treatment withp-nitrophenyl trifluoroacetate or trifluoroacetic anhydride, or byconversion to the 2-trimethylsilylethyl carbamate by the method ofCarpino or the FMOC group by the method of Carpino and Han.

3. Protection of thiols. The mercapto group is reacted by the method ofArmitage, et al, to produce the S-disulfide.

4. Protection of alcohols. Alcohols may be protected by reaction withbenzoyl chloride or a substituted benzoyl chloride, eg, anisoylchloride, in pyridine to form the ester. Alternatively, the hydroxylgroup is silylated with t-butyldiphenylsilyl chloride and imidazole inDMF.

B. General procedure for formation of the morpholino ring.

1. Using ammonia. The furanoside (1 mole) is oxidized in methanol (4L)at room temperature by the addition of sodium periodate (1.1 mol) in 400mL of warm water with vigorous stirring and exclusion of light. Afterthe oxidation is complete, the reaction is filtered to remove sodiumiodate and the filtrate treated with ammonium biborate (1.2equivalents). After stirring for 30 minutes, the mixture is treated withsodium cyanoborohydride (1 mol) followed by 6N hydrochloric acid until apH=4.5 is obtained. The reaction is allowed to sit at zero degrees C.overnight and then evaporated.

As an alternative, p-toluenesulfonic acid (or other arylsulfonic acid)may be used in place of 6N HCl in the reduction step. In certain cases acrystalline salt of the morpholino derivative and the sulfonic acid isobtained which may be filtered off and used in the next step. Thismethod is especially effective for morpholino derivatives of uridine,N-4 benzoylcytidine and N-2 phenylacetylguanosine.

2. Using primary amines. The ribofuranoside is oxidized with periodateas in the example above, and the filtered dialdehyde treated with 1.2moles of a primary amine. Two amines which are satisfactory for thispurpose are 4-methoxyaniline and 4-methoxybenzyl amine. After stirringfor 30 minutes, the mixture is treated with sodium cyanoborohydride (1mol) followed by 6N hydrochloric acid until a pH=4.5 is obtained. Thereaction is allowed to sit at room temperature overnight and the productN-aryl or N-alkyl morpholino subunit is filtered from solution.

C. General procedure for protection of the morpholino ring nitrogen.

The crude residue, or sulfonate salt, from the morpholino ring synthesisis suspended in DMF (2L) and treated with triethylamine (10 moles) andevaporated to near dryness. The residue is again suspended in DMF (2L)and treated with triethylamine (4 moles) and trityl chloride (2 moles)while the temperature is maintained at 10 degrees C. The reaction isvigorously stirred for 15 minutes at room temperature, then quenched bythe addition of piperidine (1 mole). After 5 minutes, the reaction ispoured into 20L of a one to one water/satd NaCL solution. The solids arecollected, washed with water, and dissolved in 2L of 20%methanol/chloroform. To this is added 2L of 20% isopropanol/chloroformand the mixture washed consecutively with water, 5% sodium bicarbonate,and brine. The organic layer is dried over sodium sulfate, filtered, andevaporated to provide crude N-tritylmorpholino subunit. The subunit maybe purified by silica gel chromatography.

As alternatives, base sensitive amine protecting groups may beincorporated, for example, 9-fluorenylmethylcarbonyl (using FMOCchloride in pyridine/DMF and quenching with water).

D. General procedure for the removal of alkyl and aryl groups from themorpholino nitrogen of morpholino subunits.

For morpholino subunits possessing benzylic groups at the morpholinonitrogen, the compound is hydrogenated over Pd catalyst in methanol ofmethanol/DMF mixtures. The secondary amine produced may be protected asin the general Example.

For morpholino subunits possessing either 4-methoxyphenyl or4-methoxybenzyl groups at the morpholino nitrogen, the compound isdissolved in methanol or methanol/DMF mixture containing 4 molarequivalents of acetic acid and 2 molar equivalents of sodium acetate.Cerric ammonium nitrate (2 molar equivalents) is added and the reactionstirred at room temperature for 1-24 hours. After evaporation of thesolvents the morpholino nitrogen may be protected as in the generalExample.

EXAMPLE 2 Preparation of Morpholino Subunits from SubstitutedHexopyranosides

A. General procedure for protection of the R group.

1. Protection of amino groups on heteroaromatic rings. Protection isdone as in Example 1A1.

2. Protection of aliphatic or carbocyclic aromatic amines. Protection isdone as in Example 1A2.

3. Protection of thiols. Protection is done as in Example 1A3.

4. Protection of alcohols. Protection is done as in Example 1A4.

B. General procedure for formation of the morpholino ring.

The morpholino ring is constructed as in Example 1B with the soleexception that 2.2 moles of sodium periodate are used in the oxidationstep.

C. General procedure for protection of the morpholino ring nitrogen. Themorpholino ring nitrogen is protected as in Example 1C.

D. General procedure for the removal of alkyl and aryl groups from themorpholino nitrogen of morpholino subunits.

The nitrogen is deprotected as in Example 1D.

EXAMPLE 3 Preparation of Subunits with Nucleobase Side Chains

The example illustrate the use of the D-sugars. The enantiomericsubunits may be obtained by employing the corresponding L-sugars.

A. Uracil as nucleobase.

1. Uridine was converted into the morpholino subunit 32.2b(Xi=uracil-1-yl) by the general procedure.

2. Uracil is bis-trimethylsilylated according to the procedure ofNiedballa and Vorbruggeno Uracil is dissolved in benzene, and added to1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose 33.1b (Xi=β-OAc) (Niedbalaand Vorbruggen) in 1,2 dichloroethane followed by tin tetrachloride in1,2-dichloroethane according to the method of Vorbruggen and Niedballato provide the 1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)uracil 33.1b(Xi=β-uracil-1-yl). Following methanolysis with sodium methoxide inmethanol the 1-(B-D-glucopyranosyl)uracil 33.1a (Xi=β-uracil-1-yl) isobtained. This is converted into the morpholino subunit 32.2b(Xi=β-uracil-1-yl) by the general procedure.

B. Thymine as nucleobase.

1. Ribothymidine 32.1a (Xi=β-thymin-1-yl) is prepared by the method ofTronchet, et al. It is converted into the morpholino subunit 32.2b(Xi=β-thymin-1-yl) by the general procedure.

2. Thymine is silylated by the general procedure of Wittenberg andreacted as for uracil in the Example 3a2 above to prepare the morpholinoT derivative 32.2b (Xi=β-thymin-1-yl). A wide variety of other5-substituted uracils (halo, alkynyl, alkyl, alkenyl, nitro) may beprepared in this manner. In some cases the use of acetonitrile in theHilbert-Johnson reaction is advantageous.

C. N4-benzoylcytosine as nucleobase.

1. Cytidine was converted into the morpholino subunit 32.2b(Xi=β-N4-benzoylcytidin-1-yl) by the general procedure.

2. The 1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)uracil from Example3A2 is treated with phosphorus pentasulfide in benzene to provide the4-thiouracil derivative. Reaction with triethylamine and dimethylsulfate produce the S-alkylated species which is converted into1-(β-D-glucopyranosyl)cytosine 33.1a (Xi=β-cytidin-1-yl) by treatmentwith methanolic ammonia. Following protection of the heterocyclic aminethis is converted into the morpholino subunit 32.2b(Xi=β-N4-benzoylcytidin-1-yl) by the general procedure.

3. The 1-(β-D-glucopyranosyl)uracil from Example 3A2 is treated withhexmethyldisilazane by the method of Vorbruggen, et al. Followingprotection of the heterocyclic amine this is converted into themorpholino subunit by the general procedure.

4. Cytosine is silylated by the general method of Wittenberg. It isreacted with 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose in 1,2dichloroethane followed by tin tetrachloride in 1,2-dichloroethane (oracetonitrile) according to the method of Vorbruggen and Niedballa toprovide the 1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)cytosine 33.1b(Xi=β-cytidin-1-yl). Following methanolysis with sodium methoxide inmethanol the 1-(β-D-glucopyranosyl) cytosine is obtained. Followingprotection of the heterocyclic amine this is converted into themorpholino subunit 32.2b (Xi=β-N4-benzoylcytidin-1-yl) by the generalprocedure.

D. N4-Benzoyladenine as nucleobase.

1. Adenosine was converted into the morpholino subunit 32.2b(Xi=β-N6-benzoyladenin-1-yl) by the general procedure.

2. N-6-Benzoyl-9-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)adenine(Lichtenhaler, et al.) is treated with 2:1 ammonium hydroxide/DMF at 45degrees C. for 15 hours to give 9-(β-D-glucopyranosyl)adenine 33.1a(Xi=β-adenin-9-yl). The amino group is protected as in the generalExample and the morpholino subunit 32.2b (Xi=β-N4-benzoyladenin-9-yl)produced by the general procedure for hexopyranosides. A more directmethod for the conversion of the glucoside into the morpholino subunitemploys the selective O-deacylation procedure of Rammler and Khorana onN-6-Benzoyl-9-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)adenine to giveN-6-Benzoyl-9-(β-D-glucopyranosyl)adenine which is converted into themorpholino subunit by the general procedure.

E. Hypoxanthine as nucleobase

1. Inosine was converted into the morpholino subunit 32.2b(Xi=β-hypoxanthin-9-yl) by the general procedure for ribofuranosides.

2. Inosine is silylated by the general procedure of Wittenberg. It isreacted with 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose in 1,2dichloroethane followed by tin tetrachloride in 1,2-dichloroethane (oracetonitrile) to prepare9-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)hypoxanthine 33.1b(Xi=β-hypoxanthin-9yl). Methanolysis of the esters and application ofthe standard procedure for morpholino ring synthesis produce the subunit32.2b (Xi=β-hypoxanthin-9-yl).

F. N2-Phenylacetylguanine as nucleobase.

1. Guanosine was converted into the morpholino subunit 32.2b(Xi=β-N-2-phenylacetylguanin-9-yl) by the general procedure forribofuranosides.

2. N-2-Acetyl-9-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)guanine(Lichtenhaler, et al.) was treated with 2:1 ammonium hydroxide/DMF at 45degrees C. for 15 hours. The amino group is protected as in the generalExample and the morpholino subunit 32.2b(Xi=β-N-2-phenylacetylguanin-9-yl) produced by the general procedure forhexopyranosides.

EXAMPLE 4 Preparation Subunits with Modified Nucleobase Side Chains

A variety of pyrimidines, purines, and their analogs may be convertedinto the corresponding ribofuranosides and hexopyranosides by themethods of Niedballa and Vorbruggen and by the methods of Lichtenhaler,et al. These may be further processed to morpholino subunits as per theexamples below.

A. 6-Methyluracil as side chain. 6-Methyluridine (Winkley and Morris) isconverted into the N-tritylated morpholino subunit by the generalprocedure to give 32.2b (Xi=β-6-methyluracil-1-yl).

B. N3,6-Dimethyluracil as side chain. Treatment of 32.2b(Xi=β-6-methyluracil-1-yl) from Example 4A with methyl iodide and DBU inDMF affords the N-3 methylated derivative 32.2b(Xi=β-N3,6-dimethyluracil-1-yl).

C. 6-Methylcytosine as side chain.

1. 6-Methylcytidine (Winkley and Robins) is converted into themorpholino subunit 32.2b (Xi=β-6-methylcytosin-1-yl) by the generalprocedure.

2. Treatment of 32.2b (Xi=β-6-methyluracil-1-yl) from Example 4A withTBDMS-Cl in pyidine followed by triisopropylbenzenesulfonyl chloride inmethylene chloride containing triethylamine provides the O-4 sulfonatedspecies which is converted into 32.2b(Xi=β-N4-benzoyl-6-methylcytosin-1-yl) by treatment with ammonia in DMFfollowed by protection of the base by the standard procedure and silylcleavage.

D. N4,6-Dimethylcytosine as side chain. Treatment of 32.2b(Xi=β-6-methyluracil-1-yl) from Example 4A with TBDMS-Cl in pyidinefollowed by triisopropylbenzenesulfonyl chloride in methylene chloridecontaining triethylamine provide the O-4 sulfonated species which isconverted into 32.2b (Xi=β-N4-benzoyl-N4,6-dimethylcytosin-1-yl) bytreatment with methylamine in DMF followed by protection of the base bythe standard procedure and silyl cleavage.

E. N6,N6-dimethyladenine as side chain. Inosine was converted into theN-tritylated morpholino subunit 32.2b (Xi=hypoxanthin-9-yl) by thegeneral procedure. Following conversion into the 5-t-butyldimethylsilylether using TBDMS-Cl in pyridine, treatment withtriisopropylbenzenesulfonyl chloride in methylene chloride containingtriethylamine provide the O-6 sulfonated species which is converted into32.2b (Xi=β-N6,N6-dimethyladenin-9-yl) by treatment with dimethylaminein DMF and silyl cleavage.

F. 8-Methylhypoxanthine as side chain. 8-Methylhypoxanthine (Koppel andRobins) is silylated by the general procedure of Wittenberg andconverted into8-methyl-9-(2,3,5-Tri-O-benzoyl-beta-D-ribofuranosyl)hypoxanthine 32.1b(Xi=β-8-methylhypoxanthin-9-yl) by the method of Lichtenhaler, et al.This product is converted into the morpholino subunit32.2b(Xi=β-8-methylhypoxanthin-9-yl) produced by the general procedurefor ribofuanosides.

G. 8-Methylhypoxanthine as side chain. The hypoxanthine morpholinosubunit from Example 3E is treated with 1.1 equivalents of sodiumhydride in DMF followed by methyl iodide to produce the1,8-dimethylhypoxanthine morpholino subunit 32.2b(β-N1-methylhypoxanthin- 9-yl). The use of other alkyl groups allows theformation of other 1-alkyated hypoxanthine subunits.

H. N1,8-Dimethylhypoxanthine as side chain. The 8-methylhypoxanthinemorpholino subunit from Example 4F is treated with 1.1 equivalents ofsodium hydride in DMF followed by methyl iodide to produce the1,8-dimethylhypoxanthine morpholino subunit 32.2b(β-N1,8-dimethylhypoxanthin-9-yl).

I. 8-Bromo-N2-phenylacetylguanine as side chain. The guanosine isbrominated by stirring with N-bromosuccinimide in DMF at roomtemperature by the method of Srivastava and Nagpal. This is convertedinto the morpholino subunit 32.2b(Xi=β-8-bromo-N2-phenylacetylguanin-9-yl) by protection of the amine,morpholino ring synthesis and tritylation as per the general proceudres.The 8-bromoadenine and 8-bromohypoxanthine species may be preparedsimilarly.

J. 8-Methylthio-N2-phenylacetylguanine as side chain. The 8-bromoguaninederivatives in Example 4I are converted into the 8-methylthio species byreaction with sodium thiomethoxide in DMF. The 8-bromoadenine and8-bromohypoxanthine species may be similarly converted.

EXAMPLE 5 Preparation of Subunits with Non-Nucleobase Side Chains

A. Methyl 4(5)-methylimidazole-5(4)-carboxylate as side chain.

Methyl 4(5)-methylimidazole-5(4)-carboxylate is silylated and reactedwith an equimolar amount of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranoseby the method of Cook, et al., using at least 1.44 mole of stannicchloride per mole sugar yields the acetylated sugar. Methanolysis withsodium methoxide in methanol provides methyl5-methyl-1-(β-D-glucopyranosyl)imidazole-4-carboxylate 33.1a(Xi=4-methoxycarbonyl-5-methylimadzol-1-yl. This may be converted intothe morpholino subunit 32.2b(Xi=β-4-methoxycarbonyl-5-methylimadzol-1-yl) by the standard procedure.

B. 2-Oxo-1,2-dihyropyridin-1-yl as side chain.

The pyridone ribofuranoside 32.1a (Xi=β-2-oxo-1,2-dihyropyridin-1-yl)(Pischel and Wagner) is conveted into the corresponding morpholinosubunit 32.2b (Xi=β-2-oxo-1,2-dihyropyridin-1-yl) by the standardprocedures.

C. 2-Oxo-1,2-dihyropyrimidin-1-yl as side chain.

The pyrimidone ribofuranoside 32.1a(Xi=β-2-oxo-1,2-dihyropyrimidin-1-yl) (Holy, et al) is conveted into thecorresponding morpholino subunit 32.2b(Xi=β-2-oxo-1,2-dihyropyrimidin-1-yl) by the standard procedures.

D. Benzimidazole as side chain.

The benimidazole containing ribofuranoside 32.1a(Xi=β-benzimidazol-1-yl) prepared by the method of Southon andPfleiderer is converted into the morpholino subunit 32.2b(Xi=β-benzimidazol-1-yl) by the standard procedure.

E. Phenyl as side chain.

The methods below may be used to prepare a wide variety of arylsubstutitued morpholino subunits.

1. 34.1a (Xi=C₆ H₅) The C-phenyl glycoside 33.1a (Xi=β-phenyl) preparedby the method of Czernecki and Ville is converted into the morpholinosubunit 32.2b (Xi=β-phenyl) by the general procedure.

2. 34.2a (Xi=C₆ H₅) 3,4,6-Tri-O-benzyl-1,2-anhydro-β-D-mannopyranose wasreacted with lithium diphenyl cuprate (Posner) using the procedure ofBellosta and Czernecki. The phenyl mannopyranoside 33.3d (Xi=β-phenyl)is hydrogenated to remove the benzyl groups and converted into themorpholino subunit by the standard procedure to provide 34.2a(Xi=α-phenyl).

3. 34.3a (Xi=C₆ H₅) This is made from L-mannose by the procedure in 4E2.

4. 34.4a (Xi=C₆ H₅) This is made from L-glucose by the procedure in 4E1.

F. Alkyl as side chain.

The methods below may be used to prepare a wide variety of alkyl oraralkyl (for example, benzyl or phenethyl) substutitued morpholinosubunits.

1. 34.1a (Xi=CH₃) The C-methyl glycoside 33.1b (Xi=β-methyl) prepared bythe method of Bellosta and Czernecki is converted into the morpholinosubunit 32.2b (Xi=β-methyl) by the general procedure.

2. 34.2a (Xi=CH₃) 3,4,6-Tri-O-benzyl-1,2-anhydro-β-D-mannopyranose wasreacted with lithium dimethyl cuprate (Posner) using the procedure ofBellosta and Czernecki. The methyl mannopyranoside 33.3d (Xi=α-ethyl) ishydrogenated to remove the benzyl groups and converted into themorpholino subunit by the standard procedure to provide 34.2a(Xi=α-methyl).

3. 34.3a (Xi=CH₃) This is made from L-mannose by the procedure in 4E2.

4. 34.4a (Xi=CH₃) This is made from L-glucose by the procedure in 4E1.

G. Aliphatic side chains bearing bearing hydroxy groups.

1. Hydroxymethyl.

a. 34.1a (Xi=CH₂ OTBDPS): The vinyl glucopyranoside 33.1d(Xi=β-ethenyl), prepared by the method of Kraus and Molina is ozonolyzedin a mixture of methanol and ethyl acetate and after removal of theozone the reaction is quenched with sodium borohydride in methanol toprovide the alcohol 33.1d (Xi=β-CH₂ OH). This is converted into thet-butyldiphenylsilyl ether by the general procedure. Catalytichydrogenation removes the benzyl protecting groups and the molecule isconverted into the morpholino subunit 34.1a [32.2b] (Xi=β-CH₂ OTBDPS) bythe general procedure.

b. 34.2a (Xi=CH₂ OTBDPS):3,4,6-Tri-O-benzyl-1,2-anhydro-β-D-mannopyranose is reacted with lithiumdivinyl cuprate (Posner) (REF) using the procedure of Bellosta andCzernecki. The vinyl mannopyranoside 33.3d (Xi=β-ethenyl) is furthertreated as per the procedure in Example 4G1a to provide 34.2a (Xi=CH₂OTBDPS).

c. 34.3a (Xi=CH₂ OTBDPS): The alcohol 33.1d (Xi=β-CH₂ OH) prepared as inExample 4G1a is converted into the t-butyldiphenylsilyl ether by thegeneral procedure. Catalytic hydrogenation removes the benzyl groups.The glcoside is converted to the "α-morpholino" subunit 34.3a (Xi=CH₂OTBDPS) by the procedure in Summerton, et al (U.S. Pat. No. 5,235,033).This compound may also be prepared by application of the procedures inExample 4G1b to L-mannose.

d. 34.4a (Xi=CH₂ OTBDPS): The hydroxymethyl glucopyranoside 33.1d(Xi=β-CH₂ OH) prepared as in Example 4G1a is reacted withtrimethylacetyl chloride in pyridine. Catalytic hydrogenation removesthe benzyl protecting groups and the molecule is converted into themorpholino subunit 32.2b (Xi=β-CH₂ OCO-C(CH₃)₃) by the generalprocedure. The free alcohol is silylated according to the generalprocedure and the product is treated with lithium aluminum hydride inTHF to prepare the free alcohol 34.4a (Xi=CH₂ OTBDPS). This compound mayalso be obtained by application of the procedures in Example 4G1a toL-glucose.

2. Hydroxyethyl.

a. 34.1 (Y=CH₂ OH, Xi=CH₂ CH₂ OTBDPS): The tetrabenzylated hydroxyethylderivative 33.1d (Xi=β-CH₂ CH₂ OH) prepared by the method of Allevi, etal, is protected as the t-butyldiphenylsilyl ether by the generalprocedure, the benzyl groups removed by catalytic hydrogenation over Pdon charcoal, and the morpholino ring formed by the general procedure.Alternatively, the vinyl glucopyranoside 33.1d (Xi=β-ethenyl) fromExample 4G1a is treated with borane-THF followed by alkalinehydroperoxide to yield the tetrabenzylated starting material.

b. 34.2 (Y=CH₂ OH, Xi=CH₂ CH₂ OTBDPS): The tetrabenzylated hydroxyethylderivative 33.1d (Xi=α-CH₂ CH₂ OH), prepared by the method of Allevi, etal, is protected as the t-butyldiphenylsilyl ether by the generalprocedure, the benzyl groups removed by catalytic hydrogenation over Pdon charcoal, and the morpholino ring formed by the general procedure.Alternatively, the vinyl mannopyranoside 33.3d (Xi=α-ethenyl) fromExample 4G1b is reacted with sodium hydride in DMF with benzyl chloride,then treated with borane-THF followed by alkaline hydroperxide to yieldthe 33.1d (Xi=αCH₂ CH₂ OH). This is protected as thet-butyldiphenylsilyl ether by the general procedure, the benzyl groupsremoved by catalytic hydrogenation over Pd on charcoal, and themorpholino ring formed by the general procedure.

c. 34.3 (Y=CH₂ OH, Xi=CH₂ CH₂ OTBDPS): Is prepared from L-glucose orL-mannose by the methods in Example 4G2b.

d. 34.4 (Y=CH₂ OH, Xi=CH₂ CH₂ OTBDPS): Is prepared from L-glucose by themethods in Example 4G2a.

3. Hydroxypropyl.

a. 34.1 (Y=CH₂ OH, Xi=CH₂ CH₂ CH₂ OTBDPS): Xanthate 32.1 (R₂,R₃=isopropylidene, R₅ =benzoyl, Xi=O-CS-SCH₃) (Araki, et al. ) is reactedwith methyl acrylate in the presence of tributyltin hydride and 2,2'-azobis (isobutyronitrile) as initiator to produce the C-riboside ester32.1 (R₂,R₃ =isopropylidene, R₅ =benzoyl, Xi=CH₂ CH₂ CO₂ CH₃). Treatmentwith methanolic HCl gave the free triol which was converted into themorpholino subunit 32.2b (Xi=β-CH₂ CH₂ CO₂ CH₃) by the standardprocedure. Protection of the hydroxy group as the benzyloxymethyl ether(Stork and Isobe) followed by treatment with lithium aluminum hydride inTHF, then silylation as in the standard procedure, and catalytichydrogenation provides the desired protected alcohol 34.1 (Y=CH₂ OH,Xi=CH₂ CH₂ CH₂ OTBDPS).

b. 34.2 (Y=CH₂ OH, Xi=CH₂ CH₂ CH₂ OTBDPS): Ester 33.1b (Xi=α-CH₂ CH₂ CO₂CH₃) (Adlington, et al.) is reacted with methanolic HCl to give the freetetraol which was converted into the morpholino subunit 32.2b (Xi=α-CH₂CH₂ CO₂ CH₃) by the standard procedure. Protection of the hydroxy groupas the benzyloxymethyl ether (Stork and Isobe) followed by treatmentwith lithium aluminum hydride in THF, then silylation as in the standardprocedure, and catalytic hydrogenation provides the desired protectedalcohol 34.2 (Y=CH₂ OH, Xi=CH₂ CH₂ CH₂ OTBDPS).

a. 34.3 (Y=CH₂ OH, Xi=CH₂ CH₂ CH₂ OTBDPS): Is prepared from L-glucose bythe methods in Example 4G3b.

a. 34.4 (Y=CH₂ OH, Xi=CH₂ CH₂ CH₂ OTBDPS): Is prepared from L-ribose bythe methods in Example 4G3a.

4. Homologous ω-hydroxyalkyl derivatives. Higher order alcohols34.1-34.4 (Y=CH₂ OH, Xi=CH₂ [CH₂ ]_(n) CH₂ OTBDPS) may be made by thefollowing procedure from lower order alcohols: The hydroxymethyl groupof 34.1-34.4 (y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS) is protected as thebenzyloxymethyl ether by the method of Stork and Isobe, then the TBDPSgroup is removed with tetrabutylammonium fluoride. The alcohol isconverted into a tosylate and reacted with sodiodiethylmalonate in DMSO.The ester is saponified, acidified and decarboxylated. Followingtreatment with trityl chloride in DMF to replace any trityl cleaved inthe process, the acid is reduced with lithium aluminum hydride in THF,the alcohol silylated by the standard procedure to provide 34.1-34.4(Y=CH₂ OH, Xi=[CH₂ ]_(n+2) OTBDPS).

5. Other alcohol containing side chains. The hydroxyymethyl group of34.1-34.4 (Y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS) is protected as thebenzyloxymethyl ether by the method of Stork and Isobe, then the TBDPSgroup is removed with tetrabutylammonium fluoride. The alcohol isconverted into an aldehyde with diisopropylcarbodiimide in DMSOcontaining a small amount of pyridinium methanesulfonate. The aldehydemay be reacted with any of a large variety of Grignard or organolithiumreagents to provide secondary alcohols. These may be silylated by thegeneral procedure and the hydroxymethyl freed by hydrogenolysis. Thesecondary alcohols may by oxidized to ketones which may be reacted againwith any of a large variety of Grignard or organolithium reagents toprovide tertiary alcohols. These typically do not require protection andthe final subunit may be prepared by hydrogenolysis.

H. Aliphatic side chains containing carbon-carbon double bonds. Thehydroxyymethyl group of 34.1-34.4 (Y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS) isprotected as the benzoate ester then the TBDPS group is removed withtetrabutylammonium fluoride. The alcohol is converted into the aldehydewith diisopropylcarbodiimide in DMSO containing a small amount ofpyridinium methanesulfonate. The aldehyde may be reacted with any of alarge variety of Wittig reagents (Maercker) to produce alkenes. Forexample, reaction of 34.1 (Y=CH₂ OBz, Xi=CHO) with isopropylidenetriphenylphosphorane (prepared from isopropyltriphenylphosphoniumbromide and butyllithium in THF) provides the alkene 34.1 (Y=CH₂ OBz,Xi=CH=C(CH₃)₂). This is converted into the morpholino subunit 34.1(Y=CH₂ OH, Xi=CH=C(CH₃)₂) by saponification.

Similarly, reaction with benzylidene triphenylphosphorane (prepared frombenzyltriphenylphosphonium chloride and butyllithium in THF) followed bysaponification provides the morpholino subunit 34.1 (Y=CH₂ OH,Xi=CH=CHPhenyl), the isomers of which may be separated by silica gelchromatography.

Subunits 34.2-34.4 with aliphatic side chains containing carbon-carbondouble bonds are prepared in a similar fashion.

I. Aliphatic side chains containing carboxylic acids and esters.

Alcohol 34.1 (Y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS) is treated with 80% aceticacid in water followed by reaction with benzyl chloroformate to providethe carbamate 34.1c (Xi=[CH₂ ]_(n) OTBDPS). The alcohol is protected asthe benzyloxymethyl ether by the method of Stork and Isobe, then theTBDPS group is removed with tetrabutylammonium fluoride. Thehydroxymethyl group is converted into a carboxyl by oxidation withpotassium permanganate or pyridinium dichromate. The acids are protectedby conversion into the ester by treatment with diisopropylcarbodiimide,4-dimethylaminopyridine and either methyl alcohol or2-(phenylsulfonyl)ethyl alcohol in dichloromethane. Treatment withhydrogen and Pd on charcoal followed by tritylation of the morpholinonitrogen prepares the morpholino subunits.

Similar procedures may be performed on the acids 34.1-34.4 (Y=CH₂ OH,Xi=[CH₂ ]_(n) OTBDPS) to prepare the corresponding acid. Other alcoholsmay be employed to produce a large variety of esters.

J. Aliphatic side chains containing carboxylic acid amides. The acidsprepared in part I above are reacted with diisopropylcarbodiimide andmorpholino in dichloromethane to produce the morpholino amide.Conversion to the subunit follows hydrogenolytic cleavage of thecarbamate and acetal with Pd on charcoal. Other amides may be preparedby use of ammonia or other amines.

K. Aliphatic side chains containing amines.

1. From alcohol derivatives. The hydroxyymethyl group of 34.1-34.4(Y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS) is protected as the benzyloxymethylether by the method of Stork and Isobe, then the TBDPS group is removedwith tetrabutylammonium fluoride. The alcohol is converted into analdehyde with diisopropylcarbodiimide in DMSO containing a small amountof pyridinium methanesulfonate. The alcohol is treated withtriphenylphosphine, sodium or lithium azide and carbon tetrabromide inDMF to produce the azide by the method of Yamamato, et al. Catalyticreduction with Pd on charcoal in the presence of ammonia provides theamine and frees the alcohol. The amine is protected as the generalprocedure.

2. From alcohols via oxidation to the aldehyde and reductive amination.The hydroxyymethyl group of 34.1-34.4 (y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS)is protected as the benzyloxymethyl ether by the method of Stork andIsobe, then the TBDPS group is removed with tetrabutylammonium fluoride.The alcohol is converted into an aldehyde with diisopropylcarbodiimidein DMSO containing a small amount of pyridinium methanesulfonate. Thealdehyde is treated with a large excess of the appropriate amine inmethanol at pH=6 in the presence of sodium cyanoborohydride. Theresulting amine is protected as in the general Examples and the acetalcleaved by hydrogenolysis. Additionally, the ketones prepared in Example4G5 may be employed as starting materials for the production of moreamines species.

L. Preparation of thiol derivatives.

The hydroxyymethyl group of 34.1-34.4 (Y=CH₂ OH, Xi=[CH₂ ]_(n) OTBDPS)is protected as the benzoate ester then the TBDPS group is removed withtetrabutylammonium fluoride. The alcohol is converted into the tosylateby treatment with p-toluenesulfonyl chloride in pyridine. The tosylategroup is displaced with thiourea to give the thiol which is protected byconversion into the S-ethyl disulfide by the general method. Thebenzoate group is removed by saponification.

M. Hydrogen as side chain.

1. (S)-4-Trityl-2-hydroxymethylmorpholino 32.2b (Xi=H) is prepared fromthe (S)-4-tert-butoxycarbonyl-2-hydroxymethylmorpholino prepared byYanagisawa and Kanazaki, by cleavage of the Boc group in 50%trifluoroacteic acid in dichloromethane followed by retritylation by thegeneral method.

2. (R)-4-Trityl-2-hydroxymethylmorpholine 32.2b (Xi=H) is prepared fromthe (R)-4-tert-butoxycarbonyl-2-hydroxymethylmorpholine prepared byYanagisawa and Kanazaki, by cleavage of the Boc group in 50%trifluoroacteic acid in dichloromethane followed by retritylation by thegeneral method.

EXAMPLE 6 Synthetic Strategies for Stereochemical Control of R and YGroups of Morpholino Subunits

Examples 3 and 4 above generally illustrate the preparation ofmorpholino subunits with nucleobase or modified nucleobase side chainswith groups R and Y in FIG. 6 both in the β position (FIG. 34.1). Thisderives principally from the use of D-glucose or D-galactose or D-riboseand their derivatives as precursors. For the preparation of theenantiomeric subunits, that is, morpholino subunits with groups R and Yin FIG. 6 both in the α position (species 34.4), corresponding L-sugarsare employed.

For the preparation of morpholino subunits with nucleobase or modifiednucleobase side chains with groups Y in FIG. 6 in the β position, andgroup R in the α position (species 34.3), it is preferred to use themethod of Chow and Danishefsky. In this procedure silylated nucleobasesand modified nucleobases are reacted with3,4,6-tri-O-TBDMS-1,2-anhydro-β-D-mannopyranose to give theO-TBDMS-α-D-glycosides which are converted into the morpholino subunits34.3 following desilylation with tertabutylammonium fluoride. For thepreparation of morpholino subunits with nucleobase or modifiednucleobase side chains with groups Y in FIG. 6 in the α position, andgroup R in the β position (species 34.4), it is preferred to use themethod of Chow and Danishefsky. In this procedure silylated nucleobasesand modified nucleobases are reacted with3,4,6-tri-O-TBDMS-1,2-anhydro-β-L-mannopyranose to give theO-TBDMS-α-L-glycosides which are converted into the morpholino subunits34.4 following desilylation with tertabutylammonium fluoride.

EXAMPLE 7 Preparartion of Morpholino Subunits with Representative Y andZ

A. 7.1 (Xi=β-N4-benzoylcytosin-1-yl).

The alcohol 32.2b (Xi=β-N4-benzoylcytosin-1-yl) is oxidized to thealdehyde 7.1 (Xi=β-N4-benzoylcytosin-1-yl) with diisopropyl carbodiimidein DMSO containing a small amount of pyridinium methanesulfonate.

B. 7.2 (X=OH, Xi=β-N4-benzoylcytosin-1-yl).

The aldehyde from Example 7A is oxidized with potassium permanganate inacetone or t-butanol/dioxane/water buffered with magnesium sulfate tothe acid 7.2 (X=OH, Xi=β-N4-benzoylcytosin-1-yl). Higher yields may beobtained if the trityl group in the alcohol 32.2b(Xi=β-N4-benzoylcytosin-1-yl) is replaced with a CBz group. Oxidationwith permanganate, hydrogenolysis of the CBz group, and retritylationprepare the acid needed for activation.

C. 7.2 (X=O-p-nitrophenyl, Xi=β-N4-benzoylcytosin-1yl).

The acid from Example 7B is reacted with p-nitrophenol and diisopropylcarbodiimide in dichloromethane to produce the ester 7.2(X=O-p-nitrophenyl, Xi=β-N4-benzoylcytosin-1-yl) suitable for couplingreactions.

D. 7.2 (X=imidazol-1-yl, Xi=β-N4-benzoylcytosin-1yl).

Reaction of the acid with carbonyl diimidazole produces the imidazolide7.2 (X=imidazol-1-yl, Xi=β-N 4-benzoylcytosin-1-yl) suitable forcoupling reactions.

E. 7.3 (X=Cl, Xi=β-N6-benzoyladenin-1-yl).

The alcohol 32.2b (Xi=β-N6-benzoyladenin-1-yl) is converted into thetosylate using tosyl chloride and pyridine. This is reacted withthiourea in methanol to provide the thiol deriviative. This may beoxidized to the sulfonic acid using potassium permanganate in acetone ort-butanol/dioxane/water buffered with magnesium sulfate. Higher yieldsin the oxidation are obtained if the trityl group is replaced by thebenzyloxycarbonyl group. The sulfonic acid is isolated as itstriethylamonium salt by extraction into chloroform from water saturatedwith triethylamine hydrochloride. The salts of sulfonic acids can beeasily chromatographed on silica gel usingtriethylamine/methanol/chloroform mixtures if the silica is firstpre-eluted with 2% triethylamine in chloroform. Retritylation may beefected by hydrogenolysis with Pd on charcoal to remove the carbamatefollowed by tritylation by the general procedure. For activation, tenmmole of the triethylamine salt of sulfonate subunit is dissolved in 10ml of dichloromethane and then 40 mmole of pyridine is added. Thissolution is chilled for 15 minutes on a bed of dry ice and then 11 mmoleof phosgene (20% in Toluene) is slowly added while the solution israpidly stirred. After addition the solution is allowed to come to roomtemperature and then washed with aqueous NaHCO₃, dried, andchromatographed on silica gel eluting with a mixture of chloroform andacetone to give the desired sulfonyl chloride 7.3 (X=Cl,Xi=β-N-6-benzoyladenin-1-yl).

F. 7.4 (X=OH, Xi=α-methyl).

The alcohol 32.2b (Xi=α-methyl) is oxidized to the aldehyde 7.1(Xi=α-methyl) with diisopropyl carbodiimide in DMSO containing a smallamount of pyridinium methanesulfonate. This is reacted with2,6-dithianylidene-triphenylphosphorane by the method of Kruse, et al.The resulting ketenedithioacetal is converted into the carboxylic acidby hydrolysis with mercuric chloride in wet acetonitrile to give thesubunit 7.4 (X=OH, Xi=α-methyl), which can be chromatographed on silicagel using triethylamine/methanol/chloroform mixtures if the silica isfirst pre-eluted with 2% triethylamine in chloroform.

G. 7.4 (X=O-p-nitrophenyl, Xi=α-methyl).

The acid salt from the previous example is activated by treatment withdiisopropylcarbodiimide in dichloromethane containing p-nitrophenolcontaining 1 equivalent of pyridinium p-toluenesulfonate.

H. 7.5 (X=OH, Xi=α-methyl).

Benzyl α-bromoacetate is reacted with triphenylphosphine and thephosphonium salt product is reacted with sodium hydroxide to produce theylid. This is reacted with aldehyde 7.1 (Xi=α-methyl), produced fromalcohol 32.2b Xi=α-methyl) as in Example 7A, to give the unsaturatedester. Treatment with hydrogen and Pd on charcoal yields the acid 7.5(X=OH, Xi=α-methyl), which can be chromatographed on silica gel usingtriethylamine/methanol/chloroform mixtures if the silica is firstpre-eluted with 2% triethylamine in chloroform.

I. 7.5 (X=O-p-nitrophenyl, Xi=α-methyl).

The acid salt from the previous example is activated by treatment withdiisopropylcarbodiimide in dichloromethane containing p-nitrophenolcontaining 1 equivalent of pyridinium p-toluenesulfonate.

J. 7.6 (X=O-p-nitrophenyl, Y=O, Xi=β-N2-phenylacetylguanin-9-yl).

Dry, N-protected, 5'-hydroxyl morpholino subunit 32.2b(Xi=β-N2-phenylacetylguanin-9-yl) (1 mmol), is treated withbis-(p-nitrophenyl)carbonate (BNPC) and triethylamine (TEA) in DMF underanhydrous conditions. The solution is stirred for three hours, thenevaporated to dryness. The residue is dissolved in chloroform andchromatographed on silica gel eluting with a chloroform/methanol mixtureto give activated subunit.

K. 7.6 (X=imidazol-1-yl, Y=S, Xi=β-N2-phenylacetylguanin-9-yl).

Dry, N-protected, 5'-hydroxyl morpholino subunit 32.2b(Xi=β-N2-phenylacetylguanin-9-yl) is treated withthiocarbonyldiimidazole in pyridine at room temperature for 12 hours.Water is added to quench the reagents, the solvents evaporated and theresidue is dissolved in chloroform and chromatographed on silica geleluting with an a chloroform/methanol mixture to give activated subunit.

L. 7.7 (X=Cl, Xi=β-CH₂ CH₂ OTBDPS).

An N-tritylated morpholino subunit 32.2b (Xi=β-CH₂ CH₂ OTBDPS) isdetritylated by treatment with 2% dichloroacetic acid in dichloromethanefollowed by addition to ether to precipitate the product salt. The crudesalt is dissolved in dichloromethane/pyridine and treated with 3equivalents of dimethoxytrityl chloride. The solvents are evaporated andthe residue taken up in 1:1 methanol/acetic acid to cleave the DMT groupon the nitrogen. The solvents are removed, the residue dissolved indichloromethane, washed with water, sodium bicarb solution and brine.The solution is dried over sodium sulfate, filtered and evaporated togive a residue which is purified by chromatography on silica gel elutingwith a chloroform/methanol mixture. The free morphline is sulfated bytreatment with SO₃ /pyridine complex (with excess pyridine) indimethylformamide (DMF). It should be mentioned that the salts ofsulfamic acids can be easily chromatographed on silica gel usingtriethylamine/methanol/chloroform mixtures if the silica is firstpre-eluted with 2% triethylamine in chloroform. For activation, tenmmole of the triethylamine salt of sulfated subunit is dissolved in 10ml of dichloromethane and then 40 mmole of pyridine is added. Thissolution is chilled for 15 minutes on a bed of dry ice and then 11 mmoleof phosgene (20% in Toluene) is slowly added while the solution israpidly stirred. After addition the solution is allowed to come to roomtemperature and then washed with aqueous NaHCO₃, dried, andchromatographed on silica gel eluting with a mixture of chloroform andacetone to give the desired sulfamoyl chloride 7.7 (X= Cl,Xi=CH2CH2OTBDPS).

M. 7.8 (R'=H, X=Cl, Xi=β-uracil-1-yl).

The alcohol derivative 32.2b (Xi=β-uracil-1-yl) is treated withtriphenylphosphine, sodium azide and carbon tetrabromide in DMF toproduce the azide by the method of Yamamato, et al. This may be reducedby either triphenylphosphine and ammonia, or catalytic hydrogenationover Pd and charcoal. The amine is sulfated by treatment with S0₃/pyridine complex (with excess pyridine) in dimethylformamide (DMF). Itshould be mentioned that the salts of sulfamic acids can be easilychromatographed on silica gel using triethylamine/methanol/chloroformmixtures if the silica is first pre-eluted with 2% triethylamine inchloroform.

For activation, ten mmole of the triethylamine salt of sulfated subunitis dissolved in 10 ml of dichloromethane and then 40m mole of pyridineis added, This solution is chilled for 15 minutes on a bed of dry iceand then 11 mmole of phosgene (20% in Toluene) is slowly added while thesolution is rapidly stirred. After addition the solution is allowed tocome to room temperature and then washed with aqueous NaHCO₃, dried, andchromatographed on silica gel eluting with a mixture of chloroform andacetone to give the desired sulfamoyl chloride.

N. 7.8 (R'=CH₃, X=Cl, Xi=β-uracil-1-yl).

The alcohol derivative 32.2b (Xi=β-uracil-1-yl) is oxidized to thealdehyde with diisopropyl carbodiimide in DMSO containing a small amountof pyridinium methanesulfonate. The aldehyde may be reacted withmetylamine in buffered (p-nitrophenol) methanol at pH=7 in the presenceof sodium cyanoborohydride to give the morpholine-2-methanaminederivative. The amine is sulfated by treatment with S0₃ /pyridinecomplex (with excess pyridine) in dimethylformamide (DMF). It should bementioned that the salts of sulfamic acids can be easily chromatographedon silica gel using triethylamine/methanol/chloroform mixtures if thesilica is first pre-eluted with 2% triethylamine in chloroform.

For activation, ten mmole of the triethylamine salt of sulfated subunitis dissolved in 10 ml of dichloromethane and then 40 mmole of pyridineis added. This solution is chilled for 15 minutes on a bed of dry iceand then 11 mmole of phosgene (20% in Toluene) is slowly added while thesolution is rapidly stirred. After addition the solution is allowed tocome to room temperature and then washed with aqueous NaHCO₃, dried, andchromatographed on silica gel eluting with a mixture of chloroform andacetone to give the desired sulfamoyl chloride.

O. 7.9 (X=Cl, Y=O, Z=N(CH₃)₂, Xi=β-phenyl).

One mmole of 5'-hydroxyl subunit 34.1a (Xi=phenyl), protected andtritylated on the morpholino nitrogen is dissolved in 5 ml ofdichloromethane. Six mmole of N,N-diethylaniline and 2 mmole ofdimethylaminodichlorophosphate (OP(Cl)₂ N(CH₃)₂) is added to thesolution followed by the addition of 0.5 mmole of eitherN-methylimidazole, tetrazole, or 4-methoxypyridine-N-oxide. After thereaction is complete (assessed by thin layer chromatography) thereaction solution may be washed with aqueous NaH₂ PO₄. The activatedsubunit is isolated by chromatography on silica gel developed withacetone/chloroform or ethyl acetate/dichloromethane mixtures.Alternatively, the reaction mixture is placed on the top of a silicacolumn and chormatographed without workup. Thedimethylaminodichlorophosphate used in the above procedure was preparedas follows: a suspension containing 0.1 mole of dimethylaminehydrochloride in 0.2 mole of phosphorous oxychloride was refluxed for 12hours and then distilled (boiling point is 36° C. at 0.5 mm Hg).

P. 7.9 (X=Cl, Y=S, Z=O-ethyl, Xi=β-phenyl).

One mmole of 5'-hydroxyl subunit 34.1a (Xi=β-phenyl) is reacted withethyl dichlorothiophosphate according to the conditions in Example 7O.

Q. 7.10 (X=p-nitrophenyl, Xi=β-N4-benzoylcytosin-1-yl).

1. The N-tritylated morpholino subunit derivative 32.2b(Xi=β-N4-benzoylcytosin-1-yl) is detritylated using 2% acetic acid in20% trifluoroethanol/dichloromethane. The resulting secondary amine isreacted with isoamyl nitrite and the N-nitroso species reduced withhydrogen over Pd on charcoal or Zn in acetic acid. The amino group maybe protected as the benzhydryl carbamate by the method of Hiskey andAdams or as the Boc carbamate using ditertbutyl dicarbonate. The alcoholis activated for coupling as the carbonate by reaction withbis-(p-nitrophenyl) carbonate to give 7.10 (X=p-nitrophenyl,Xi=β-N4-benzoylcytosin-1-yl). Other activated species may be preparedusing thiocarbonyldiimidazole or N,N-dimethylaminophosphoryl chloride asdescribed in the preceeding examples.

2. Alternatively, ribofuranoside 32.1a (Xi=β-N4-benzoylcytosin-1-yl) isreacted with periodate as in the general method for morpholino subunitsynthesis, but t-butylcarbazate is substituted for ammonia in thereductive ring closure step to give the 5'-free subunit which may beactivated as in the example above.

R. 7.11 (X=OH, Xi=β-uracil-1-yl). The alcohol 32.2b (Xi=β-uracil-1-yl)(1 mol) is treated with an excess of sodium hydride in a DMF/THFmixture. Sodium chloroacetate (1 mol) is added and the solution stirredfor 24 hours. The solution is filtered in an inert atmosphere, andexcess of triethylammonium hydrochloride in DMF is added, the mixturefiltered, and the solvents removed by evaporation. The residue can bechromatographed on silica gel using triethylamine/methanol/chloroformmixtures if the silica is first pre-eluted with 2% triethylamine inchloroform.

S. 7.11 (X=O-p-nitrophenyl, Xi=β-uracil-1-yl).

The acid salt from the previous example is activated by treatment withdiisopropylcarbodiimide in dichloromethane containing p-nitrophenolcontaining 1 equivalent of pyridinium p-toluenesulfonate.

T. 7.12 (X=O-p-nitrophenyl, Xi=β-thymin-1-yl).

1. The subunit 32.2b (Xi=Xi=β-thymin-1-yl) is detritylated using 2%acetic acid in 20% trifluoroethanol/dichloromethane. The resultingsecondary amine is reacted with benzyl bromoacetate. The alcohol isconverted into the primary amine my the procedure in method 7M. Theamine is tritylated by the general procedure, the benzyl ester cleavedby catalytic hydrogenolysis in DMF/ethanol containing trieylamine. Theacid is activated by treatment with diisopropylcarbodiimide indichloromethane containing p-nitrophenol and one equivalent ofpyridinium tosylate.

2. Alternatively, ribothymidine (Tronchet) is reacted with periodate asin the general method for morpholino subunit synthesis, but glycinebenzyl ester is substituted for ammonia in the reductive ring closurestep to give the 5'-free subunit which may be further converted as inthe example above.

EXAMPLE 8 Representative Subunits which are Converted to MorpholinoStructures during Oligomer Assembly

A. 5'-Aminoribofuranosides 8.1.

Ribofuranosides may be converted into their 5'-amino derivatives byreaction with triphenylphosphine, sodium or lithium azide and carbontetrabromide in DMF (Yamamato), followed by reduction with eithertriphenyl phosphine/ammonia or with hydrogen over Pd on charcoal.

B. 6'-Aminohexopyranosides 8.4.

Hexopyranosides may be converted into their 6'-amino derivatives by theprocedure in Example 8A or by the following procedure. The glycoside istreated with dimethoxytrityl chloride in pyridine to selectively protectthe primary alcohol. The remaining hydroxy groups are protected byreaction with t-butyldimethylsilyl chloride in DMF containing imidazole.The dimethoxytrityl group is cleaved by treatment with zinc bromide innitromethane at room temperature (Koster, et al). The free 6'-alcohol isconverted into the 6'-amino derivative by reaction withtriphenylphosphine, sodium or lithium azide and carbon tetrabromide inDMF (Yamamato), followed by reduction with either triphenylphosphine/ammonia or with hydrogen over Pd on charcoal. The silyl groupsare removed by treatment with HF/pyridine of tetrabutylammonium fluoridein THF.

C. 5'-O-Aminoribofuranosides 8.2.

Ribofuranosides may be converted into their 5'-O-amino derivatives bythe following procedure. The glycoside is treated with dimethoxytritylchloride in pyridine to selectively protect the primary alcohol. Theremaining hydroxy groups are protected by reaction witht-butyldimethylsilyl chloride in DMF containing imidazole. Thedimethoxytrityl group is cleaved by treatment with zinc bromide innitromethane at room temperature (Koster, et al) and the primary alcoholconverted into the desired aminoxy species using N-hydroxyphthalimide bythe procedure of Vassuer, et al. The silyl groups are removed bytreatment with HF/pyridine of tetrabutylammonium fluoride in THF.

D. 5'-O-Aminohexopyranosides 8.5.

May be converted into their 6'-O-amino derivatives by the procedure forthe ribofuranosides in Example 8C.

EXAMPLE 9 Coupling Morpholino Subunits to form RepresentativeOne-Atom-Length Intersubunit Linkages and Two-Atom-Length IntersubunitLinkages

A. General.

Whenever the morpholino nitrogen of a subunit, or the terminal subunitin an oligomer, contains an acid labile group such as the trityl group,deprotection is performed with mild acid. Representative acid mixtureswhich are suitable include 10% cyanoacetic acid in 4:1dichloromethane/acetonitrile, 7% formic acid in dichloromethane, and2.5% cyanoacetic acid in 7:93 trifluoroethanol/dichloromethane. Formolecules which contain a Boc or benzhydryl carbamate a 20-50% solutionof trifluoroacetic acid in dichloromethane may by employed. The acid isremoved by precipitation of the deprotected subunit in ether if thereaction is done in homogeneous solution, or by washing with theappropriate rinse solvent if solid phase methods are employed.

Whenever the morpholino nitrogen of a subunit, or the terminal subunitin an oligomer, contains a base labile group such as the FMOC group,deprotection is performed with mild base. Representative base reagentswhich are suitable include 1-10% DBU/DMF, 10% N-methylpyrrolidine/DMF,and 2-20% piperidine/DMF. The excess reagent, dibenzofulvene, andderived by products are removed by precipitation of the deprotectedsubunit in ether if the reaction is done in homogeneous solution, or bywashing with the appropriate rinse solvent if solid phase methods areemployed. Coupling to a morpholino subunit requires that the morpholinonitrogen be present in the uncharged state.

This may be achieved as follows. A mild base such as triethylamine,diisopropylethylamine, or diisopropylaminoethanol (or its ethers oresters) is employed to neutralize residual charge produced in acidicdeprotections and/or to maintain any unreacted morpholino nitrogen inthe neutral state during the coupling reaction.

B. Coupling to form amide linkages.

The nitrophenyl ester formed in Example 7C, is dissolved in DMF or NMP(containing 0.2-0.4 molar of an appropriate base, such as methyldiisopropylaminoethyl ether) at a concentration of about 0.2 molar, andmixed with the deprotected monomeric or oliogomeric species with anuncharged morpholino nitrogen produced as in Example 9A.

C. Coupling to form an amine linkage.

The aldehyde formed in Example 7A is dissolved in methanol, orDMF/methanol mix containing nitrophenol and sodium cyanoborohydride atpH=6.5. This is mixed with the deprotected monomeric or oliogomericspecies with an uncharged morpholino nitrogen produced as in Example 9A.

1. Formation of amide linkages. The nitrophenyl ester formed in Example7G, is dissolved in DMF or NMP (containing 0.2-0.4 molar of anappropriate base, such as methyl diisopropylaminoethyl ether) at aconcentration of about 0.2 molar, and mixed with the deprotectedmonomeric or oliogomeric species with an uncharged morpholino nitrogenproduced as in Example 9A.)

EXAMPLE 10 Method for Conversion of Non-morpholino Subunit to MorpholinoSubunit during Oligomer Assembly

Oligomer may be assembled by construction of the morpholino ring from adialdehyde and a primary amine. The coupling is performed as follows.The 5'-aminoribofuranoside or 6'-aminohexopyranoside from Example 8 isprotected on the amine with trityl as in the general procedure. Themolecule is dissolved or suspended in methanol and treated withperiodate as per the general procedure in Examples 1 or 2. To thedialdehyde so formed is added a second 5'-aminoribofuranoside or6'-aminohexopyranoside followed by sodium cyanoborohydride and the pH ismaintained between 4.5 and 6.5.

A particularly advantageous method for the synthesis of oligomers bythis method involves fixing the the amino group of the firstaminoglycoside to a solid support by a cleavable anchor, as in Example16 below, and performing the oxidation and reductive amination steps onthe solid support.

EXAMPLE 11 Coupling Morpholino Subunits to form RepresentativeThree-atom-length Intersubunit Linkages

A. Formation of amide linkages.

The nitrophenyl ester formed in Example 7I, is dissolved in DMF or NMP(containing 0.2-0.4 molar of an appropriate base, such as methyldiisopropylaminoethyl ether) at a concentration of about 0.2 molar, andmixed with the deprotected monomeric or oliogomeric species with anuncharged morpholino nitrogen produced as in Example 9A.

B. Formation of carbamate linakges.

This linkage is prepared from the nitrophenyl carbonate formed inExample 7J and the morpholino-deprotected subunits/oligomer formed as inExample 9A. The coupling follows the method of Summerton and Weller(U.S. Pat. No. 5,034,506).

C. Formation of sulfamide linkages.

This linkage is prepared from the sulfamoyl chlorides produced inExamples 7M or 7N and the morpholino deprotected subunits/oligomerformed as in Example 9A. The coupling follows the method of Summertonand Weller (U.S. Pat. No. 5,034,506).

D. Formation of phosphorodiamidate linkages.

This linkage is prepared from the phosphoryl chloride produced inExample 70 and the morpholino deprotected subunits/oligomer formed as inExample 9A. The coupling follows the method of Summerton and Weller(U.S. Pat. No. 5,185,444).

EXAMPLE 12 Coupling Morpholino Subunits to form RepresentativeFour-Atom-Length Intersubunit Linkages

A. Formation of amide linkages.

1. The nitrophenyl ester formed in Example 7S, is dissolved in DMF orNMP (containing 0.2-0.4 molar of an appropriate base, such as methyldiisopropylaminoethyl ether) at a concentration of about 0.2 molar, andmixed with the deprotected monomeric or oliogomeric species with anuncharged morpholino nitrogen produced as in Example 9A.

2. The nitrophenyl ester formed in Example 7T, is dissolved in DMF orNMP (containing 0.2-0.4 molar of an appropriate base, such as methyldiisopropylaminoethyl ether) at a concentration of about 0.2 molar, andmixed with the deprotected monomeric or oliogomeric species with anuncharged morpholino nitrogen produced as in Example 9A.

B. Formation of carbazates.

This linkage is prepared from the nitrophenyl carbonate formed inExample 7Q is dissolved in DMF or NMP (containing 0.2-0.4 molar of anappropriate base, such as methyl diisopropylaminoethyl ether) at aconcentration of about 0.2 molar, and mixed with the deprotectedmonomeric or oliogomeric species with an uncharged morpholino nitrogenproduced as in Example 9A.

EXAMPLE 13 Preparation of In-Line Branches

A. From Diethylenetriamine

The triamine is reacted with one equivalent of triamine and theterminally reacted monotritylated species isolated by chromatography onalumina. The diamine is now reacted with FMOC chloride, followedimmediately by sulfation in pyridine with the sulfur trioxide/pyridinecomplex. It should be mentioned that the salts of the sulfamic acids canbe easily chromotographed on silica gel usingtriethylamine/methanol/chloroform mixtures if the silica is firstpre-eluted with 2% triethylamine in chloroform. For activation, tenmmole of the triethylamine salt of sulfated subunit is dissolved in 10ml of dichloromethane and then 40 mmole of pyridine is added. Thissolution is chilled for 15 minutes on a bed of dry ice and then 11 mmoleof phosgene (20$ in Toluene) is slowly added while the solution israpidly stirred. After addition the solution is allowed to come to roomtemperature and then washed with aqueous NaHCO₃, dried, andchromatographed on silica gel eluting with a mixture of chloroform andacetone to give the desired sulfamoyl chloride. The sulfamate can becoupled to an amino group or morpholino amine in the same fashion inwhich an activated subunit may be coupled (Examples 9-12). Followingincorporation of the branching subunit, cleavage of the trityl groupallows construction of the oligomeric branches by sequential coupling ofsubunits. When the first branch is complete is capped and the moleculetreated with 10% DBU/DMF to remove the FMOC group. The second branch maynow be synthesized using by sequential coupling of subunits.

B. From 1,3-Diamino-2-Hydroxypropane

The diamine is dissolved in DMF and treated with one equivalent oftrityl chloride. The monotritylated species is separated bychromatography on silica gel and then reacted with FMOC chloride toprotect the remaining amino group. The alcohol is reacted withbis(p-nitrophenyl carbonate) in DMF containing triethylamine to producethe activated carbonate. The carbonate can be coupled to an amino groupor morpholino amine in the same fashion in which an activated subunitmay be coupled (Example 9-12). Following incorporation of the branchingsubunit, cleavage of the trityl group allows construction of theoligomeric branch by sequential coupling of subunits. When the firstbranch is complete, it is capped and the molecule treated with 10%DBU/DMF to remove the FMOC group. The second branch may now besynthesized using by sequential coupling of subunits.

EXAMPLE 14

Preparation of Hub Branches

A. Using 1,3,5-Benzenetricarboxylic acid amides.

One mmol of 1,3,5-benzenetricarbonyl chloride in pyridine is reactedwith 1 mmol of o-nitrobenzyl alcohol, followed by 1 mmol ofp-nitrophenethyl alcohol, and the reaction quencehed with piperidine.The desired species containing one o-nitrobenzyl ester and onep-nitrophenethyl ester is isolated by chromatography on silica gel. Thefree piperazine is coupled to activated subunits prepared in Example 7using the methods in Examples 9-12. After the coupling, the product ispurified on silica gel. Additional subunits may be introduced bydetrityalation and repetition of the coupling. Following introduction ofthe final subunit, the chain is detritylated and capped with aceticanhydride. The nitrophenethyl ester is cleaved by treatment with 10%DBU/DMF. The free acid is coupled with N-trityl piperazine preparedbelow using diisopropylcarbodiimide in dichloromethane. Sububits may beintroduced by detritylation and coupling as above. When this chain isfinished it is capped with acetic anhydride.

The o-nitrobenzyl ester is cleaved by irradiation with 320 nm light.Following coupling with N-trityl piperazine as above, sububits may beintroduced by detritylation and coupling as above.

B. Using 1,3,5-Benzenetricarboxylic acid.

One mmol of 1,3,5-benzenetricarbonyl chloride in pyridine is reactedwith 1 mmol of o-nitrobenzyl alcohol, followed by 1 mmol ofp-nitrophenethyl alcohol, and the reaction quencehed with water. Thedesired species containing one o-nitrobenzyl ester and onep-nitrophenethyl ester is isolated by chromatography on silica gel. Thefree acid is coupled to subunits or preformed oligomers, at the freemorpholino nitrogen (produced by detritylation and neutralization asdescribed in Example 9), using diisopropylcarbodiimide indichloromethane. The nitrophenethyl ester is cleaved by treatment with10% DBU/DMF. The free acid is coupled to subunits or preformedoligomers, at the free morpholino nitrogen, coupled with usingdiisopropylcarbodiimide in dichloromethane. The o-nitrobenzyl ester iscleaved by irradiation with 320 nm light. The free acid is coupled tosubunits or preformed oligomers, at the free morpholino nitrogen, usingdiisopropylcarbodiimide in dichloromethane. It should be recognized thatthe acid may be employed in solid phase synthesis by coupling to agrowing chain on a solid support. The two esters which may each beselectively deprotected are reacted sequentially with subunits oroligomers.

C. From piperazine.

N-tritylpiperazine is reacted with FMOC chloride. The trityl group isremoved by the method in Example 9, and the free piperazine nitrogenreacted with an activated subunit by the method above. As many subunitsas desired may be introduced by the method in Example 14A above.Following end capping of this chain, the FMOC group is cleaved using 10%DBU/DMF. The free piperazine nitrogen is reacted with an activatedsubunit by the method above. As many subunits as desired may beintroduced by the method in Example 14A above.

EXAMPLE 15 Joining Two Ends of an Oligomer by Covalent Linkage

Two chains of a divergent branch are constructed so as to place subunitswith Xi=[CH₂ ]_(n) -SS-ethyl at the termini. The disulfide is cleavedusing dithiothreitol in mildy basic aqueous solution. The oligomericdithiol is separated from the reagents by passage over a column ofchromatographic grade polypropylene and eluting with an acetonitrile indilute aqueous acetic acid. The dilute solution is neutralized to pH=8,and treated with iodine to produce the disulfide.

EXAMPLE 16 Preparation of Oligomer Library on a Solid Support

A. Solid support

The following supports are suitable for solid phase synthesis ofoligomers: aminomethyl polystyrene resin, 1% divinylbenzene crosslinked,200-400 mesh, 0.5-1.5 mmoles N per gram (Sigma Chemical CO. A1160);polystyrene resin, 1% divinylbenzene crosslinked, grafted withpolyethylene glycol, primary amino terminated, 0.1-0.3 mmoles N per gram(TentaGel, Rapp Polymere, Germany); custom-synthesized macroporouspolystyrene, 8% divinylbenzene crosslinked, functionalized with1,12-diaminododecane, with particle sizes in the range of 50-80 micronsin diameter, and with pore sizes approximately 700 Å.

B. Construction of anchors

The following anchors are employed for solid phase oligomer synthesis:

1. 10 mmol of bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone (Pierce,Rockford Ill.) is treated with 5 mmol of momotritylpiperazine (made asin the example below) and the product purified by silica chromatographyor crystallization.

2. 2,2'-Thiodiethanol is treated with an equimolar amount ofbis(p-nitrophenyl) carbonate in DMF containing triethylamine. Themonoesterified species is purified by chromatography and reacted with anexcess of monotritylpiperazine, formed by adding trityl chloride to asolution of excess piperazine in DMF. This is converted to thenitrophenyl carbonate with a slight excess of as abovebis(p-nitrophenyl) carbonate in DMF containing triethylamine.

3. 4-Hydroxymethyl-3-nitrobenzoic acid (Knieb-Cordonier, et al) isesterifed with methanol and diisopropylcarbodiimide in dichloromethane.The alcohol is converted into the p-nitrophenylcarbonate by the methodabove and reacted with monotritylpiperazine. The ester is cleaved bysaponification and converted into the p-nitrophenyl ester by treatmentwith p-nitrophenol and diisopropylcarbodiimide in dichloromethane.

C. Construction of tethers.

Polyethylene glycol 4600 is converted into its mono-p-nitrophenylcarbonate by reaction of 10 mmol of the PEG with 1 mmol ofbis(p-nitrophenyl) carbonate in DMF containing triethylamine. To thesolution is add an excess of piperazine and the excess reagentsthoroughly removed by evaporation. The mixture is taken up in water,acidified and the amine is purified from the neutral PEG chains by ionexchange chromatography on Dowex-50. The amine is converted into theN-trityl species by the standard procedure and then the alcohol isconverted to the nitrophenyl carbonate with a slight excess ofbis(p-nitrophenyl) carbonate in DMF containing triethylamine.

D. Configuration of the particle for oligomer assembly

1. Atttachment of the tether. The solid support is placed in a columnsuch as the 2 mL polypropylene Biorad Bio-Spin Disposable ChromatographyColumn, cat #732-6008, whose frit has been replaced with a new frit(Isolab Quik-Sep Disc #D-4301). The resin is treated with DMF for 1-12hours, during which time it is placed in a aspirator vacuum to removetrapped air, and gently agitated to break up clumps. The tether isdissolved in DMF or NMP (containing 0.2-0.4 molar of an appropriatebase, such as triethylamine) at a concentration of about 0.05 molar andadded to the support. Sufficient tether is added so as to react 5- 50%of the free amines on the resin surface. After 12-48 hours at 45 degreesC. the solvents are drained and the resin washed with DMF.

2. Capping with dansyl chloride. The resin from the previous example iswashed with dichloromethane and treated with a solution of 0.2 molardansyl chloride in 20% tetramethylene sulfone in dichloromethanecontaining 0.4 molar of an appropriate amine such asdiisopropylethylamine. After 30 minutes at room temperature the resin isdrained and washed with DMF.

3. Attachment of the anchor. The trityl group is removed from the end ofthe tether by three washings with 2% dichloroacetic acid indichloromethane. The resin is washed with dichloromethane, then 5%diisopropylethylamine in dichloromethane. The anchor is dissolved in DMFor NMP (containing 0.2-0.4 molar of an appropriate base, such as methyldiisopropylaminoethyl ether) at a concentration of about 0.2 molar, andmixed with the resin containing the deprotected tether for 2-48 hours atroom temperature. The resin is thoroughly washed with DMF.

E. Solid phase synthesis of oligomers for solution phase testing.

1. Coupling to produce an oligomer with only morpholino backbone type.Aminomethyl polystyrene resin is loaded with the anchor from Example16B1 to achieve about 350 umol of trityl species per gram resin. Thefollowing cycle is repeated. Suitable washes of dichloromethane, 25%isopropanol/dichlromethane, or DMF are incorporated between the steps toremove excess reagents and reaction byproducts.

a. The protecting group is removed by an acidic reagent from Example 9A.

b. The resin is neutralized with 5-20% diisopropylethylamine indichloromethane.

c. A mixture of activated subunits prepared in Example 7 is coupled tothe end of the growing chain by the procedures defined in Examples 9-12.It is critical, in order to achieve roughly equimolar amounts of theindividual oligomers, that the concentrations of each activated subunitin the reaction be adjusted so that the rate of coupling for eachsubunit will be as nearly the same as possible. The coupling rates forthe activated subunit are determined in solution, by reaction with amonomeric morpholino subunit. The appearance of coupling product as afunction of time monitored by HPLC. A rate constant is calculated andused to adjust the concentrations in the coupling mixture so that therate of incorporation of each activated species in the mixture is thesame.

2. Preparation of a mixed backbone oligomer. The method of Example 16E1is employed, but activated subunits other than morpholino species areemployed. For example, Boc-alanine may be converted into isp-nitrophenyl ester using diisopropylcarbodiimide in dichloromethane. Itis used as an activated subunit in the repetitive steps outlined in themethod of example 16E1. The oligomer so produced has a mixedmorpholino-peptide backbone.

F. Deprotection of the oligomers.

Deprotection of the functional groups on the side chains is acheived asfollows. Silylated groups are removed by treatment with eithert-butylammonium fluoride in THF or pyridinium/HF complex in pyridine.Amides may be cleaved by ammonolysis with 2:1 conc ammonia/DMF.Phenylsulfonyl or FMOC carbamates are cleaved DBU/DMF treatment.Disulfides are converted into thiols by treatment with mercaptoethanolor dithiothreitol in DMF or water containing triethylamine.

EXAMPLE 17 Solid Phase Synthesis of Oligomers for Oligomer FamilyTesting Methods

The method of Example 16E1 is employed with the following differences. Amacroporous resin is treated with tether, then anchor. Two additionalsteps, a and e below, are incorporated into each cycle to produce thefollowing sequence:

a. The resin is distributed, in equal portions, into a number ofsynthesis columns that is the same as the number of subunits speciesdesired to couple in step e.

b. The protecting group is removed by an acidic reagent from Example 9A.

c. The resin is neutralized with 5-20% diisopropylethylamine indichloromethane.

d. Only a single activated subunit species is coupled in each column.

e. The resin is recombined.

For example, to prepare an oligomer family which consists of activatedsubunits derived from the following set of subunits:

1. 32.2b (Xi=β-methyl)

2. 32.2b (Xi=β-uracil-1-yl)

3. 32.2b (Xi=β-N4-benzoylcytosin-1-yl)

4. 32.2b (Xi=β-N6-benzoyladenin-1-l)

5. 32.2b (Xi=β-N2-phenylacetylguanin-1-yl)

requires that following incorporation of the anchor onto the resin, themacroporous resin is divided into five equal portions and placed in fivecolumns (Example 16D1) suitable for solid phase synthesis.

The synthesis cycle is then preformed with each column receiving asingle activated subunit species (prepared by the methods in Example 7,from the subunits in the list above) for the coupling step. When thecoupling step is finished, the resin is recombined and distributed intofive new solid phase synthesis columns for the second synthesis cycle,where again, each column recieves a single activated subunit species.

2. Incorporation of truncated species into the oligomer family.

a. By use of partially pre-capped subunits. The five subunits speciesfrom Example 16F1 are converted into their acetamides ortrifluoroacetamides by removal of the trityl protecting group andreaction with either 5% acetic anhydride and 5% triethylamine in DMF for5 minutes or with p-nitrophenyl trifluoroacetate in DMF.

These are then individually activated by the methods in Example 7 andindividually mixed with the corresponding activated, but stilltritylated, subunits with the same Xi group. The correct proportion ofcapped to tritylated subunits for a given synthesis cycle in theconstruction of a hexamer is given in Table 1. The synthesis is thenperformed exactly as described in Example 16F1 with these five mixturesof capped and tritylated activated subunit species.

b. By capping during the synthesis. After the deprotection step of eachcoupling cycle, the resin containing the detritylated chain is treatedwith 7% formic acid in dichloromethane. The extent of formylation iscontrolled by the length of the treatment. For example, to achieve a2.5% conversion to formylated chains requires one hour with thisreagent. As an alternative, the cleavage of trityl may be done withformic acid/dichloromethane mixtures as described in example 9A. Insteadof immediately washing the resin after detritylation, to remove theacidic reagent, the reaction is continued to promote the formylation ofthe morpholino nitrogen.

EXAMPLE 18 Determination of Oligomer Sequence

A. Removal of oligomers from a Selected Bead

The treament necessary to remove the oligomer from the resin depends onthe anchor:

1. Anchor from 16B1 is cleaved by treatment with 10% DBU/DMF.

2. Anchor from 16B2 is cleaved by treatment with mercaptoethanol ordithiothreitol in DMF or water containing triethylamine.

3. Anchor from 16B3 is cleaved by iradiation of the resin with light of350 nm. Wavelengths shorter than 300 nm are are excluded by a pyrexfilter.

B. Analysis of the oligomer by mass spectrometry.

A single bead, containing an oligomer family, and sorted by theprocedures described above, is washed by the methods described above toremove protein. The anchor is then cleaved by the method in Example 16Husing 2 uL of reaction solution. The reaction mixture is combined with amixture of sinapinic acid and aqueous acetonitrile (4 parts). Thesolution is then introduced onto the probe of a Matrix Assisted LaserDesorption Time Of Flight (MALDE-TOF) mass spectrometer. The compositionand sequence of the oligomer is determined by the molecular weights ofthe peaks of the full-length molecule and the truncated species.

EXAMPLE 19

Density Gradient Separation of Oligomer-Library Particles

Libraries of oligomers are formed on the particles in accordance withthe examples above. The oligomer-particles preferably contain an intensedye or fluorescent material to facilitate visualization of individualparticles. Each particles is preferably prepared to contain a singlefamily of N-subunit oligomer species, and together the collection ofparticles in a given preparation contain the full library of oligomerspecies.

To utilize such a library of oligomer-particles for detection of targetbinding by one or more component oligomer species and for determiningthe sequence of an oligomer family containing an oligomer which exhibitssaid target binding, the oligomer-particle library preparation is mixedwith a suitable concentration of target in a solution having a densitygreater than that of the oligomer-particle, but less than that of anoligomer-particle/target complex. Sucrose solutions are generallyconvenient for this purpose. After gentle mixing for a period of timesufficient to allow binding of target to any particle-bound oligomerwhich has a suitable affinity for said target, the solution is allowedto stand for a period of time, whereupon any oligomer-particle/targetcomplex which forms will settle to the bottom of the container. If theparticles are quite small (eg., 20 to 30 microns in diameter) or thebuoyant density differential between the solution and theoligomer-particle/target is small, then centrifugation can be used tospeed the settling of complexed particles.

Alternatively, the oligomer-particle preparation is mixed with asuitable concentration of target in a solution containing adensity-gradient-forming component, such as metrizamide, Centrifugationin an ultracentrifuge then generally rapidly separatesoligomer-particles from any oligomer-particle/target complex which mayhave formed.

Although the invention has been described with reference to specifcsynthetic, and sequencing methods, it will be appreciated that variouschanges and modification can be made without departing from theinvention.

It is claimed:
 1. A combinatorial library of oligomers, each formed ofat least four linked morpholino subunits of the form: ##STR4## in which(i) morpholino subunit structures are linked together by linkages L oneto four atoms long joining the morpholino nitrogen of one subunit to the4' cyclic carbon of an adjacent subunit, (ii) X_(i) is a side chain insubunit i in each oligomer of the library, (iii) the different oligomersin the library have different sequences of side chains in at least threesubunit positions, (iv) X_(i) is selected from the group consisting ofpurines, pyrimidines, non-nucleobase aromatic side chains, aliphaticside chains, and mixed aromatic/aliphatic moieties, and (v) said librarycontains at least 1,000 different side chain sequence oligomers.
 2. Thecomposition of claim 1, wherein oligomer linkages in the library includeone-atom linkages of the form: ##STR5## where X_(i+1) is a side chain ina subunit adjacent subunit i.
 3. The composition of claim 1, whereinoligomer linkages in the library include carbonyl-containing linkages ofthe form: ##STR6##
 4. The composition of claim 1, wherein an oligomer inthe library includes side chains selected from the group consisting ofpurines and pyrimidines, and side chains selected from the groupconsisting of a non-nucleobase aromatic moieties, aliphatic moieties,and mixed aromatic/aliphatic moieties.
 5. The composition of claim 1,wherein said oligomers are effective to hybridize, by Watson-Crick basepairing to random-sequence oligonucleotides.
 6. The composition of claim1, wherein said oligomers have different sequences of linkages.
 7. Thecomposition of claim 1, wherein the linkages are selected from the groupconsisting of 3-atom carbamate and 3-atom phosphorodiamidate.
 8. Thecomposition of claim 1, wherein the oligomers include an oligomer havingone or more morpholino subunit structures that are covalently attachedto a linkage that itself directly links two additional morpholinosubunit structures in the oligomer.
 9. The composition of claim 1,wherein the combinatorial library is formed on a plurality of particles,each particle having a surface coating of oligomer molecules of the samesequence.
 10. The composition of claim 9, wherein the oligomer moleculeson each particle are carried on dendritic polymers attached to theparticles and coupled to the oligomer molecules through cleavablelinkages.
 11. The composition of claim 9, wherein the particles aremacroreticular particles having selected sizes in the 40-200 μm range,and the oligomer molecules are coupled to the particles throughcleavable linkages.
 12. A composition of oligomers, each formed of atleast four linked morpholino subunits of the form: ##STR7## in which (i)morpholino subunit structures are linked together by one-atom linkagesjoining the morpholino nitrogen of one subunit to the 4' cyclic carbonof an adjacent subunit, (ii) X_(i) is a side chain in subunit i in eacholigomer of the composition, (iii) the different oligomers in thecomposition have different sequences of side chains in at least threesubunit positions, and (iv) X_(i) is selected from the group consistingof purines, pyrimidines, non-nucleobase aromatic side chains, aliphaticside chains, and mixed aromatic/aliphatic moieties.
 13. A composition ofoligomers, each formed of at least four linked morpholino subunits ofthe form: ##STR8## in which (i) morpholino structures are linkedtogether by carbanoyl-containing linkages joining the morpholinonitrogen of one subunit to the 4' cyclic carbon of an adjacent subunit,(ii) X_(i) is a side chain in subunit i in each oligomer of thecomposition, (iii) the different oligomers in the composition havedifferent sequences of side chains in at least three subunit positions,and (iv) X_(i) is selected from the group consisting of purines,pyrimidines, non-nucleobase aromatic side chains, aliphatic side chains,and mixed aromatic/aliphatic moieties.
 14. The composition of claim 13,wherein X_(i) is a purine or pyrimidine Watson-Crick base-pairingmoiety.